Cryopreservation of bioprinted tissue fibers

Abstract

Aspects of the disclosure include cryopreserved tissue fibers and multilayer structures comprising said fibers, wherein the fibers encapsulate dissociated cells, and where the fibers and / or structured are cryopreserved prior to aggregation of the encapsulated cell population. Methods of making and using such fibers and structures are also provided.

Description

CRYOPRESERVATION OF BIOPRINTED TISSUE FIBERSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 548,164, filed November 10, 2023, the contents of which are incorporated herein by reference in their entirety.FIELD OF DISCLOSURE

[0002] The present invention relates to cryopreserved tissue fibers encapsulating cell populations with improved cell viability and, optionally, increased cell density, and to methods of making and using same.BACKGROUND OF THE DISCLOSURE

[0003] Implantable solid organ cell therapies generally require 5% to 20% of the native epithelial (parenchymal) cell mass to be replaced with transplanted cells in order to establish a sufficient threshold of function needed for therapeutic effect. To further improve the longevity and function of these cells, many treatments focus on mimicking organ density by using cell aggregation. This increases cell to cell attachments, essential to maintain epithelial cell types which in turn results in increased physiologically relevant function. At the same time, however, this increased reliance on cell aggregation results in other, significant complications.

[0004] As but one example, cell therapies can be difficult to produce and bank due to their lack of functional stability outside of an in vivo environment. In the absence of cryopreservation they must be used within a very short period of time after production, a factor severely limiting the availability and scalability of this technical approach. Unfortunately, the effective cry opreservation of “solid” spheroids or aggregates in particular has not been fully demonstrated, and is currently not achievable on a large enough scale. Traditional methods of cryopreserving spheroids often result in significant cell death and reduced function. (Acker, et al., Cryobiology 1999 Jun; 38(4):363-71; Acker, et al., Biophys J 2001 Sep; 81 (3): 1389-1397).

[0005] Moreover, intracellular ice crystal formation must be avoided to prevent lysis of the cells via use of specialized cryoprotectant. A permeabilization solution such as dimethylsulfoxide (DMSO) is generally used to permeabilize the cells and reduce cellular water content, thereby reducing ice crystal formation that can damage the cells. In this regard, recent bioprinting efforts have incorporated DMSO directly into the bioprinted materials in an effort to ensure adequate access and coverage. Lee et al. Innovative Cryopreservation Process Using a Modified Core / Shell Cell-Printing with a Microfluidic System for Cell-Laden Scaffolds, ACS Applied Materials & Interfaces (2018). This necessarily increases the exposure of the cells to DMSO, however, which recent reports indicate can fundamentally damage the cells. See Verheijen et al. DMSO induces drastic changes in human cellular processes and epigenetic landscape in vitro. Sci Rep 9, 4641 (2019). As such, DMSO is clearly not inert and including such in the bioprinting process is particularly ill-advised.

[0006] Accordingly, improvements in design, materials and techniques are still needed to provide tissue therapies that can be effectively cryopreserved while retaining sufficient cellular viability and functionality.SUMMARY OF DISCLOSURE

[0007] The present invention successfully resolves the foregoing challenges in the art by providing cryopreserved core / shell tissue fibers, medical devices comprising same, and methods of making and using same. Aspects include a cryopreserved tissue fiber comprising a continuously bioprinted core / shell fiber encapsulating a cell population for implantation, the core-shell fiber comprising a core and at least one shell, the cell population comprising at least 90%, at least 95%, at least 98%, at least 99%, or 100% dissociated cells embedded in a biocompatible matrix, where the fiber is cryopreserved prior to aggregation of the dissociated cells.

[0008] The dissociated cells may be encapsulated in the core and / or in the at least one shell. In embodiments, the dissociated cells are encapsulated in the core. In embodiments, the dissociated cells are encapsulated in the at least one shell. In embodiments, the core / shell fiber comprises at least two shells, the innermost shell comprising an annulus layer encapsulating the cell population.

[0009] In embodiments, the continuously bioprinted core / shell fiber comprises a core and at least one shell, optionally wherein the core has a material strength less than that of the shell. In embodiments, the core and the at least one shell comprise the same hydrogel material. Preferably,the hydrogel material is alginate. In embodiments, the core and / or the at least one shell comprise a chemically modified alginate.

[0010] In embodiments, the biocompatible matrix further comprises a viscosifier, preferably where the viscosifier comprises fish gelatin. In embodiments, the viscosifier is selected from the group comprising or consisting of hydroxypropylmethylcellulose (HPMC), fish gelatin (e.g. low or high molecular weight), polyvinyl alcohol (PVA, e g. 90 kDA PVA), polyethylene glycol (PEG), non-fish gelatin, a poloxamer such as Pluronic (e.g. F126 or F68), xantham gum, gellan gum, guar gum, gum arabic, or combinations thereof. In embodiments, the viscosifer is present in an amount of about 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, or 5% of the biocompatible matrix. In embodiments, the viscosifier is hydroxypropylmethylcellulose (HPMC), fish gelatin, non-fish gelatin, poly(ethylene glycol) (PEG), guar gum, gum arabic, xanthan gum, or polyvinyl alcohol (PVA, e.g. 90 kDA PVA). In embodiments, the HPMC is about 1, 1.25% or 1.5% of the biocompatible matrix, the fish gelatin is about 4, 5 or 6% of the biocompatible matrix, or the PVA is about 1, 2 or 3% of the biocompatible matrix.

[0011] In embodiments, the biocompatible matrix does not include DMSO. In embodiments, the core / shell fiber is bioprinted without including DMSO in the core or in the at least one shell.

[0012] In embodiments, the cell population comprises primary cells from endocrine and exocrine glands selected from the group consisting of pancreas, liver, kidney, parathyroid, thyroid, pineal gland, pituitary gland, thymus, adrenal gland, ovary, testis, tonsillar, enteroendocrine cells, stem cells, stem-cell derived cells of any of the foregoing primary cell types, or cells engineered to secrete a therapeutic agent. In embodiments, the dissociated cells comprise HepG2s, primary human hepatocytes (PHHs), mesenchymal stem cells (MSCs), embryonic stem cell- or induced pluripotent stem cell-derived hepatocyte-like cells, liver stem cell-derived hepatocyte-like cells, xenogeneic hepatocytes, genetically engineered stem cell-derived hepatocytes, stem-derived or primary endothelial cells, and combinations thereof. In embodiments, the dissociated cells comprise HepG2s, primary human hepatocytes (PHHs), and / or mesenchymal stem cells (MSCs), and combinations thereof. In embodiments, the dissociated cells comprise a 3: 1 ratio of PHHs and MSCs. In some embodiments, the dissociated cells comprise adrenal cells.

[0013] In another aspect, a composition / medical device is provided for implanting a cell population into a subject in need thereof, comprising a multilayer structure comprising acontinuously bioprinted tissue fiber according to any embodiments of the present disclosure, wherein the multilayer structure is cryopreserved prior to aggregation of the dissociated cells. In embodiments, the composition / medical device comprises a multilayer lattice structure as described herein.

[0014] In embodiments, the multilayer structure comprises at least one conformal coating. In embodiments, the coating comprises a hydrogel having a material strength less than both the core and the at least one shell of the fiber. In embodiments, the core, the at least one shell, and the coating comprise the same hydrogel material; preferably wherein the hydrogel material is alginate. In embodiments, the solid core, the at least one shell, and / or the coating comprises a chemically modified alginate

[0015] In embodiments, the biocompatible matrix comprises at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, or at least about 80% v / v dissociated cells in physiological matrix material. In embodiments, the cryopreserved tissue fiber is a high-density tissue fiber and the biocompatible matrix comprises at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, or at least about 85% v / v dissociated cells in physiological matrix material. In embodiments, the biocompatible matrix comprises a ratio of cells : physiological matrix material of at least about 2: 1, preferably at least about 3: 1, more preferably at least about 4:1.

[0016] In embodiments, the cell population is encapsulated in the core, and the core diameter is between about 50 um and about 250 um or between about 100 um and about 200 um, preferably between about 130 um and about 200 um, preferably between about 140 um and about 190 um, more preferably between about 150 um to 175 um, more preferably about 150 um, about 155 um, about 160 um, about 165 um, about 170 um, or about 175 um. In embodiments, the shell thickness is between about 30 um and about 100 um or between about 20 um and about 200 um, more preferably between about 45 um and 70 um, more preferably about 60 um, about 65 um, or about 70 um. In embodiments, the total fiber diameter is between about 200 um and 500 um or between about 270 um and about 330 um, preferably between about 280 um and about 320 um, morepreferably about 290 um to about 310 um, more preferably about 290 um, about 300 um, or about 310 um.

[0017] In embodiments, the cell population is encapsulated in the annulus layer, and the core diameter is between about 50 um and 750 um or between about 300 um and about 500 um, preferably between about 350 um and about 450 um, more preferably about 350 um, about 375 um, about 400 um, about 425 um, or about 450 um. In embodiments, the annulus layer thickness is between about 50 um and about 300 um or between about 100 um and about 300 um, preferably between about 130 um and about 200 um, preferably between about 140 um and about 190 um, more preferably between about 150 um to 175 um, more preferably about 150 um, about 155 um, about 160 um, about 165 um, about 170 um, or about 175 um. In embodiments, the tissue fiber further comprises an outer shell having a thickness between about 30 um and about 120 um or between about 30 um and 80 um, more preferably between about 45 um and 70 um, more preferably about 60 um, about 65 um, or about 70 um. In embodiments, the total fiber diameter is between about 300-1400 um or between about 700-1200 um, preferably between about 800 um and about 1000 um, more preferably about 800 um, about 900 um, or about 1000 um.

[0018] In embodiments, the structure comprises at least two, three, four, or five layers formed by the continuous fiber, preferably wherein the structure comprises two layers or three layers or four layers, more preferably wherein the structure comprises four layers. In embodiments, the core and / or the at least one shell is compartmentalized along the length of the fiber.

[0019] Aspects of the invention also include a method of implanting a cell population into a subject in need thereof, comprising: a. thawing a cryopreserved tissue fiber or composition / medical device of the present disclosure; and b. implanting said fiber or said composition / medical device into said subject. In embodiments, the method can further include culturing said tissue fiber or said composition / medical device in fresh culture media after thawing until the dissociated cells form aggregates in said tissue fiber, prior to implantation into said subject.

[0020] Aspects of the invention include a method of fabricating a cryopreserved tissue fiber or an implantable multilayer structure of the present disclosure, comprising: a) providing a bioprinting system comprising a print head comprising a plurality of microfluidic channels to selectively provide a core material, at least one shell material, and a sheath fluid to a dispensingorifice; a receiving surface for receiving the materials dispensed from the orifice, a positioning unit for positioning the orifice of the print head in three dimensional space, the positioning unit operably coupled to the print head, and at least one dispensing means for dispensing the fiber from the dispensing orifice; b) via the bioprinting system, simultaneously printing the core and the at least one shell of the tissue fiber, wherein the cell population is encapsulated in the core and / or in the at least one shell; and c) cryopreserving said bioprinted tissue fiber with a cryoprotectant; where the cell population comprises at least 95% dissociated cells embedded in a biocompatible matrix, and wherein the fiber is cryopreserved prior to aggregation of the dissociated cells; preferably within 15 minutes to 3 hours of bioprinting, more preferably with 30, 45 or 60 minutes of bioprinting.

[0021] In embodiments, the printing step comprises dispensing the continuously bioprinted core / shell fiber on the receiving surface to form the multilayer structure, wherein the multilayer structure is cryopreserved prior to aggregation of the dissociated cells in the fiber. In embodiments, the method further comprises coating the multilayer (e.g. lattice) structure with at least one conformal coat after printing is completed

[0022] In embodiments, said cryopreserving step comprises adding cryoprotectant to the printed fiber at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 minutes before freezing.

[0023] In embodiments, the biocompatible matrix further comprises a viscosifier, preferably wherein the viscosity of the core material and the shell material are substantially the same. In embodiments, the viscosifier is selected from the group comprising or consisting of hydroxypropylmethylcellulose (HPMC), fish gelatin (e.g. low or high molecular weight), polyvinyl alcohol (PVA, e.g. 90 kDA PVA), polyethylene glycol (PEG), non-fish gelatin, a poloxamer such as Pluronic (e.g. F126 or F68), xantham gum, gellan gum, guar gum, gum arabic, or combinations thereof. In embodiments, the viscosifer is present in an amount of about 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, or 5% of the biocompatible matrix. In embodiments, the viscosifier is hydroxypropylmethylcellulose (HPMC), fish gelatin, or polyvinyl alcohol (PVA, e.g. 90 kDA PVA). In embodiments, the HPMC is about 1, 1.25% or 1.5% of the biocompatible matrix, the fish gelatin is about 4, 5 or 6% of the biocompatible matrix, or the PVA is about 1, 2 or 3% of the biocompatible matrix. In embodiments, the core material and at least one shell material are bioprinted without DMSO.

[0024] In embodiments, said receiving surface comprises a fabrication platform for supporting the continuously bioprinted core / shell fiber, the fabrication platform comprising a frame defining a void and comprising a plurality of posts on opposing sides of the frame for securing and suspending the continuously bioprinted fiber during printing; and the printing step comprises dispensing the fiber around a plurality of said posts to form a lattice structure comprising at least two, three, four, or five layers of the fiber.

[0025] Aspects of the invention include a method of treating a subject, comprising implanting a composition / medical device of the present disclosure into the subject. In embodiments, the subject is a human subject suffering from a liver disease or condition.INCORPORATION BY REFERENCE

[0026] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.BRIEF DESCRIPTION OF THE DRAWINGS

[0027] FIGS. 1A-1C show H&E staining of pre-aggregated and solid fibers (FIG. 1A), fluorescent micrographs of a live / dead assay for solid fibers and pre-aggregated fibers at days 1, 3 and 7 post-printing (FIG. IB), and albumin levels for solid fibers and pre-aggregated fibers for two different donors (FIG. 1C).

[0028] FIGS. 1D-1G show relative albumin production (FIG. ID), albumin production (FIG. IE), a live / dead fluorescent staining image (FIG. IF), and micrographs of fibers (FIG. 1G).

[0029] FIGS. 2A-2B show fluorescent micrographs of a live / dead assay for solid fibers and pre-aggregated fibers from two different donors (FIG. 2A) and albumin and Cyp3A4 activity for solid fibers and pre-aggregated fibers from two different donors (FIG. 2B).

[0030] FIG. 3A depicts a brightfield microscope image of a fiber and a fluorescence microscopy image of a fiber depicting pre-aggregated PHH and MSC cells within the fiber, the cells can be distributed in the core of the fiber.

[0031] FTG. 3B depicts an annulus fiber printed with a celkfish gelatin ratio of 3: 1 and cell concentration of 8.2 x 106million PRHs and 4.1 x 106rat MSCs per ml, where the cells are in the first shell (annulus).

[0032] FIGS. 4A-4D show a live / dead assay (FIG. 4A), plasma albumin levels (FIG. 4B), images of surgical sites in NSG mice (FIG. 4C) and plasma albumin levels (FIG. 4D).

[0033] FIGS. 5A-5B show a brightfield image of a printed fiber (FIG. 5A) and a live / dead assay (FIG. 5B).

[0034] FIGS. 6A-6B show live / dead fluorescent staining for a HepG2 solid fiber 3 days post-thawing (FIG. 6A) and a HepG2 non -cry opreservation control solid fiber (FIG. 6B).

[0035] FIG. 7 shows Cyp3A4 activity and albumin production (FIG. 5C) for HepG2 cells 5 days post-printing.

[0036] FIGS. 8A-8C show solid fiber and aggregate annulus formats printed with PHH + MSCs or adrenocortical cells (FIGS. 8A and 8B) and a cross-sectional view of a bioprinted annulus fiber (FIG. 8C) containing dissociate PHH / MSCs.

[0037] FIG. 9 shows a representative image of a solid fiber containing single cells in an annulus layer.

[0038] FIGS. 10A-10B shows fluorescent micrographs for a live / dead assay for solid fiber or aggregate annulus formats for both fresh and cryopreserved fibers and printed with PHH + MSCs or adrenocortical cells (FIG. 10A in color; FIG. 10B in grayscale).

[0039] FIGS. 11A-11B show albumin secretion levels for solid and aggregate annulus formats printed with PHH + MSCs (FIG. 11 A) and cortisol secretion levels for solid and aggregate annulus formats printed with adrenocortical cells (FIG. 1 IB).

[0040] FIGS. 12A-12B show relative albumin secretion levels for solid and aggregate annulus formats printed with PHH + MSCs (FIG. 12A) and relative cortisol secretion levels for solid and aggregate annulus formats printed with adrenocortical cells (FIG. 12B).DETAILED DESCRIPTION

[0041] 3D bioprinting is an additive manufacturing process where synthetic tissue structures, optionally cell laden, are laid down in a layer-by-layer fashion to obtain multi-layer 3D structures. Various types of 3D bioprinting techniques have been developed, including extrusion (Panwar A et al., Molecules. (2016); 21 : 685; Sakai S et al., Biofabrication. (2018); 10: 045007; Han HW and Hsu SH, Neural Regener. Res. (2017); 12: 1595), inkjet (Gao G et al., Biotechnol. Lett. (2015); 37: 2349; Gao G and Cui X, Biotechnol. Lett. (2016); 38: 203; Bsoul A et al., Lab Chip. (2016); 16: 3351) laser assisted (Sorkio A et al., Biomaterials. (2018); 171 : 57; Pages E et al., J. Nanotechnol. Eng. Med. (2015); 6: 021006; Catros S et al., In Vivo and In Situ Biofabrication by Laser- Assisted Bioprinting, Elsevier, Winston-Salem, USA. (2015)), and stereolithographic (SLA) (Miri AK et al., Adv. Mater. (2018); 30: 1800242; Wang Z et al., ACS Appl. Mater. Interfaces. (2018); 10; 26859; Wang Z et al., Biofabrication. (2015); 7: 045009) printing methods. Of these, extrusion is one of the most common, whereby bioinks are dispensed through one or more syringes to form layer-by-layer scaffolds from fibers.

[0042] Advances have also led to the use of microfluidics-based 3D bioprinting systems (Beyer ST et al., in 2013 Transducers Eurosensors XXVII 17thInt. Conf. Solid-State Sensors, Actuators, Microsystems. IEEE, Piscataway, NJ (2013); pp.1206-1209; Beyer ST et al., in The 17thInt. Conf, on Miniaturized Systems for Chemistry and Life Sciences. (2013); pp. 176-178). With these systems and techniques, a plurality of materials (e.g., bioink, cross-linker, etc.) flow through microchannels which can allow for precision control of one or more of flow, switching, mixing, and the like. When used with a sheath flow that surrounds at least one inner material, microfluidic bioprinting can reduce shear stress during the printing process. Microfluidics-based 3D bioprinting can also advantageously allow for the intersecting of material flows as they exit independent flow paths into a single flowpath (e.g., dispensing channel), to facilitate the production of structures having a core surrounded by one or more shells.

[0043] The present disclosure provides for bioprinting of dissociated, singularized cells and cryopreservation prior to aggregation, resulting in improved cell viability and function. This correspondingly enables significant improvements in manufacturing workflows and allows for therapeutics to be manufactured in advance and banked for future use. As such, worldwide distribution is now feasible. Finally, cryopreservation enables treatment of emergency conditions.

[0044] Moreover, as demonstrated herein, a fiber printed in accordance with the present disclosure can also achieve higher cell densities by eliminating the typical pre-aggregation step employed in the prior art. Cell therapies in development generally employ cell aggregates encapsulated in a biomaterial, with the biomaterial occupying a significantly larger volume of space to that of the cells. This can lead to significant size limitations making the resulting implant difficult or impossible to use due to limited space at an appropriate surgical site. In accordance with the inventive teachings herein, cells are printed as a single cell suspension in smaller amounts of biomaterial, thus significantly reducing the size of the resulting bioprinted device.

[0045] Finally, issues including but not limited to immune protection, adequate oxygen and nutrient passage, and avoidance of FBR can be resolved in accordance with the inventive teachings herein.Definitions

[0046] For purposes of interpreting this specification, the following definitions will apply, and whenever appropriate, terms used in the singular will also include the plural and vice versa. In the event that any definition set forth conflicts with any document incorporated herein by reference, the definition set forth below shall control. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure pertains.

[0047] The term “hydrogel” as used herein refers to a composition comprising water and a network or lattice of polymer chains that are hydrophilic.

[0048] The term “sheath fluid” or “sheath solution” as used herein refers to a fluid that is used, at least in part, to envelope or “sheath” a material as the material is passing through a fluid channel. In embodiments, a sheath fluid comprises an aqueous solvent, e.g., water or glycerol. In embodiments, a sheath fluid comprises a chemical or physical cross-linking agent. Non-limiting examples of cross-linking agents include divalent cations (e.g. Ca2+, Ba2+, Sr2+, etc.), thrombin, and pH modifying chemicals, such as sodium bicarbonate.

[0049] The terms “segmented / compartmentalized” as used herein refer to a discontinuous nature of a type of material and / or a biological material included in core or shell(s) of the fibersdisclosed herein, e.g. wherein there are intentional gaps in the deposition of the type of material and / or biological material along a length of the fiber. The spacing (e.g., length) between such segments / compartments may be regular (e.g., an approximately same spacing between regions of biological material), or the spacing may be different.

[0050] The term “solid core” as used herein refers to a core of a fiber of the present disclosure that is comprised of a particular material (e.g., hydrogel cross-linkable by a chemical cross-linking agent), such that the core does not comprise a lumen along the entire length of the fiber. The term is not intended to refer to a core that is entirely impenetrable along its length, as solid cores of the present disclosure may enable the passage of particular fluids, molecules and / or ionic species throughout the core.

[0051] The term “biocompatible materials” as used herein refers to materials in which biological materials including but not limited to cells can be incorporated into and / or be in contact with said biocompatible materials, and where said biocompatible materials do not exhibit an adverse effect on the ability of the biological materials to carry out one or more functions (e.g., cellular functions including but not limited to secretion of biologically relevant molecular species, agonist / receptor binding, signal transduction, and the like).

[0052] The term “immunoprotective” as used herein refers broadly to a design aspect of a fiber of the present disclosure that serves to reduce, prevent or eliminate the host immune response including, e.g., immune cell invasion of the fiber upon implantation of the fiber into a body (e.g., mammalian body).

[0053] The term “agent” as used herein refers to any protein, nucleic acid molecule (including chemically modified nucleic acid molecules), antibody, small molecule, organic compound, inorganic compound, or other molecule of interest. An agent can include a biologically relevant agent, a therapeutic agent, a diagnostic agent, a pharmaceutical agent, a chelating agent, a crosslinking agent, etc. A therapeutic or pharmaceutical agent is one that alone or together with an additional compound induces a desired response (such as inducing a therapeutic or prophylactic effect when administered in a manner consistent with the present disclosure to a subject. A biologically relevant agent is one that supports another biological process, for example an agent that supports cell viability.

[0054] The term “cryoprotectant” as used herein refers to a composition used for cryopreservation that is intended to protect cells from damage during the cryopreservation process. In some embodiments, a cryoprotectant does not include dimethyl sulfoxide (DMSO). In some embodiments, a cryoprotectant can include DMSO. In some embodiments, a DMSO-containing cryoprotectant can include CRYOSTOR(R) CS10, CRYOSTOR(R) CS5, CROSTOR CS2, PSC Cry opreservation Kit, Synth-a-Freeze™ Cryopreservation Medium, CELLBANKER(R) 1, CELLBANKER(R) 2, and STEM-CELLBANKER(R) GMP Grade. In some embodiments, a DMSO-free cryoprotect can include CRYOSTOR(R) CSB, CrySOfree™ DMSO-free Cry opreservation Medium, STEM-CELLBANKER(R) DMSO-free, GMP Grade, Cell-Vive™ CD DMSO-Free Cry opreservation Solution.

[0055] The term “dissociated cells” as used herein refers to cell populations comprising primarily single cells and smaller clusters of cells, (e.g. less than 10, 12 or 15 cells), preferably wherein any cell clusters are smaller than 20-50 micrometers in diameter. As is known in the art, dissociated cells have typically undergone an enzymatic or mechanical digestion process to separate the cells from one another. This is in contrast to cells within a solid organ or spheroid where the cells are connected to each other either by direct cell-to-cell junctions or by being fixed together with extracellular matrix. Spheroids and / or organoids comprising more than 15 cells and / or at more than 50 um in size are not “dissociated” cell populations as contemplated herein. Dissociated cells can also be distinguished from aggregates by the presence of brighter regions between cells under phase contrast microscopy, and irregular cluster boundaries, demonstrating the lack of strong or extensive cell-cell adhesion between adjacent cells.Cryopreserved Fiber Structures and Multilayer Lattice Structures Thereof

[0056] One aspect of the present invention is directed to a cryopreserved tissue fiber comprising a continuously bioprinted core / shell fiber encapsulating a cell population for implantation, the core-shell fiber comprising a core and at least one shell, the cell population comprising at least 90%, at least 95%, at least 98%, at least 99%, or 100% dissociated cells, embedded in a biocompatible matrix, where the fiber is cryopreserved prior to aggregation of the dissociated cells.

[0057] In embodiments, the biocompatible matrix comprises at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, or at least about 80% v / v dissociated cells in a physiological matrix material.

[0058] In embodiments, high density tissue fibers are provided comprising at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, or at least about 80% v / v dissociated cells in a physiological matrix material.

[0059] In embodiments, the biocompatible matrix comprises a ratio of cells : physiological matrix material of at least about 2: 1, preferably at least about 3: 1, more preferably at least about 4: 1.

[0060] The dissociated cells may be encapsulated in the core and / or in the at least one shell. In embodiments, the dissociated cells are encapsulated in the core. In embodiments, the dissociated cells are encapsulated in the at least one shell. In embodiments, the core / shell fiber comprises at least two shells, the innermost shell comprising an annulus layer encapsulating the cell population. Fibers containing at least two shells, the innermost shell comprising an annulus layer are described, for example, in WO 2022 / 246550 which is incorporated herein by reference in its entirety.

[0061] In embodiments, the continuously bioprinted core / shell fiber comprises a core and at least one shell, optionally wherein the core has a material strength less than that of the shell. In embodiments, the core and the at least one shell comprise the same hydrogel material. Preferably, the hydrogel material is alginate. In embodiments, the core and / or the at least one shell comprise a chemically modified alginate.

[0062] In embodiments, the biocompatible matrix further comprises a viscosifier, preferably wherein the viscosifier comprises fish gelatin. In embodiments, the viscosifier can comprise hydroxypropylmethylcellulose (HPMC), fish gelatin (e.g. low or high molecular weight), polyvinyl alcohol (PVA, e.g. 90 kDA PVA), polyethylene glycol (PEG), non-fish gelatin, a poloxamer such as Pluronic (e.g. F126 or F68), xantham gum, gellan gum, guar gum, gum arabic, or combinations thereof. In embodiments, the viscosifer is present in an amount of about 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%,16%, 17%, 18%, 19%, or 20%, or any range therebetween of the biocompatible matrix. By way of example, but not limitation, PVA can be used at about 3%, HPMC can be used at about 1% or 1.25%, low molecular weight fish gelatin can be used at about 5%, PEG can be used at about 1% to about 5%, non-fish gelatin can be used at less than about 5%, poloxamers can be used at about 5% to about 10%, and gums can be used at about 1% of the (core of the fiber). In embodiments, the biocompatible matrix does not include DMSO. In embodiments, the core / shell fiber is bioprinted without including DMSO in the core or in the at least one shell.

[0063] In another aspect, a composition / medical device is provided for implanting a cell population into a subject in need thereof, comprising a multilayer structure comprising a tissue fiber according to any embodiments of the present disclosure, wherein the multilayer structure is cryopreserved prior to aggregation of the dissociated cells. In embodiments, the composition / medical device is a multilayer lattice structure as described herein.

[0064] In embodiments, the multilayer lattice structure comprises at least one conformal coating. In embodiments, the coating comprises a hydrogel having a material strength less than both the core and the at least one shell of the fiber. In embodiments, the core, the at least one shell, and the coating comprise the same hydrogel material; preferably wherein the hydrogel material is alginate. In embodiments, the solid core, the at least one shell, and / or the coating comprises a chemically modified alginate

[0065] In embodiments, the solid core comprises between about 1.2 to about 1.8% alginate, preferably about 1.5% alginate.

[0066] In embodiments, the at least one shell comprises between about 1.4% to about 3.0% alginate; preferably between about 1.5% to about 2.5% alginate; more preferably between about 1.8% and about 2.2% alginate.

[0067] In embodiments, the coating comprises between about 0.2% alginate to about 2% alginate, or between about 0.25% alginate to about 1.5% alginate; preferably between about 0.3% to about 1.0% alginate; more preferably between about 0.4% to about 0.8% alginate.

[0068] In embodiments, said at least one coating comprises a first inner coating and a second outer coating. In embodiments, the first inner coating comprises a hydrogel having a material strength greater than that of the second outer coating. In embodiments, the first inner coatingcomprises between about 1.4% to about 3.0% alginate, preferably between about 1.5% to about 2.5% alginate, more preferably between about 1.8% and about 2.2% alginate. In embodiments, the second outer coat comprises between about 0.25% alginate to about 1.5% alginate, preferably between about 0.3% alginate to about 1.0% alginate, more preferably between about 0.4% alginate to about 0.8% alginate.

[0069] In embodiments, the core and / or the at least one shell is compartmentalized along the length of the fiber.

[0070] In embodiments, a multilayer lattice structure formed from a single continuous fiber can comprise more than two layers, for example 3, 4, 5, 6, 7, 8, 9, or 10 layers, or even greater than 10 layers in some examples. In embodiments, lattice structure can be square, rectangular, oval, hexagonal, circular, and the like. Dimensions of the lattice structure comprise those suitable for implantation into a particular subject (e.g., human, dog, cat, rat, pig, etc.). In embodiments where the lattice structure is approximately square, dimensions of the structure may be between about 8 mm x 8 mm to about 150 mm x 150 mm. In embodiments where the lattice structure is of a different shape than square, area of the lattice structure can range from between about 64 mm2to about 22500 mm2, or any value there between.

[0071] In embodiments, a multilayer lattice structure of the present disclosure can include 1, 2, 3, 4, or 5 shells, and 1, 2, 3, 4, or 5 coatings. Bioprinted fibers themselves can also be coated in some examples.

[0072] In embodiments, the solid core, at least one shell and one or more coatings are comprised of biocompatible material(s). Examples of biocompatible materials relevant to the present disclosure can include but are not limited to alginate, collagen, decellularized extracellular matrices, hyaluronic acid (HA), polyethylene glycol (PEG), fibrin, gelatin, gelatin methacrylate (GEL-MA), silk, chitosan, cellulose, polycaprolactone (PCL), poly(lactic acid) (PLA), poly(oligoethylene glycol methacrylate) (POEGMA), or a combination thereof In embodiments, the solid core, at least one shell and one or more coatings are comprised of a hydrogel material. Hydrogel materials relevant to the present disclosure can include but are not limited to alginate (e.g., SLG-100 alginate), chitosan, GEL-MA, agarose, PEG, PCL, poly-L-lysine (PLL), triazole, fucoidan, poly(ethylene glycol) diacrylate (PEGDA), poly (-ethylene glycol)-tetra-acrylate (PEGTA), poly (vinyl alcohol) (PVA), Hyaluronic acid (HA), hyaluronic acid methacryloyl(HAMA), collagen, methacrylated collagen (ColMA), gelatin, gellan, fibrin (fibrinogen), and combinations thereof In some embodiments, HA can be used to enhance / increase viscosity.

[0073] In embodiments, the diameter of the solid core of a cryopreserved tissue fiber of the present disclosure is between about 100 um and about 300 um, preferably between about 130 um and about 200 um, preferably between about 140 um and about 190 um, more preferably about 150 um to 175 um, more preferably about 150 um, about 155 um, about 160 um, about 165 um, about 170 um, or about 175 um, for example about 130 um, 140 um, 150 um, 155 um, 160 um, 165 um, 170 um, 175 um, 180 um, 190 um, 200 um, or any value there between..

[0074] In embodiments, the thickness of the at least one shell of a cryopreserved tissue fiber of the present disclosure is between about 30 um and 100 um, more preferably between about 40 um and 70 um, for example about 30 um, 40 um, 50 um, 60 um, 70 um, or any value there between.

[0075] In embodiments, the core and / or shell of a continuously bioprinted fiber of the present disclosure comprises a plurality dissociated, single cells, such as, by way of example, hepatocytes or other liver cells. Devices of the present disclosure may include some appropriate number of cells, depending on the species.Infill Density

[0076] The infill density of the lattice structures of the present disclosure represents an important parameter determining diffusional flow as well as vascularization. For example, lattice structures with too high an infill density may impede / degrade diffusional flow through and / or host tissue ingrowth. Alternatively, lattice structures with too low of an infill density may be undesirable in terms of one or more of structural stability, F-F adhesion, ease of retrievability, and the like. Infill density, as discussed herein, is referred to as percent infill density of the 3D lattice structures including the one or more coating(s). A lattice structure having a completely filled fibrous structure (i.e., no spaces) would thus correspond to an infill density of 100%, whereas a lattice structure that is 90% unoccupied by any fibrous structure corresponds to an infill density of 10%.

[0077] In embodiments, the infill density of suitable multilayer lattice structures of the present disclosure is between about 10% and about 90%, for example between about 20% and about 80%, for example between about 30% and about 70%, for example between about 40% and about 60%,about 30%, about 40%, about 50%, about 60%, about 70%, or about 80%, or any value there between. Preferably, the infill density is between about 50% and about 60%.

[0078] For multilayer (e.g. lattice) structures of the present disclosure, infill density is advantageously controlled via the manner in which the structures are fabricated. Specifically, as explained in more detail in International PCT Application No. PCT / CA2023 / 050667, a continuous fiber can be precisely printed into a lattice of 2, 3, 4, 5, or more layers using the fabrication platform described therein. To generate the lattice, a continuous fiber can be bioprinted around a plurality of posts that serve to impart tension to the fiber as it is being printed, which advantageously maintains the fiber in each row / column a uniform distance away from the fiber corresponding to adjacent row / column in terms of each layer, and ensuring linearity of the fiber between each opposing post as the lattice is being fabricated.

[0079] Following the fabrication of the lattice structure, one or more conformal coatings can be applied to the entirety of the exposed surfaces of the lattice structure, without disrupting the structure, and even prior to (or during) the effective fiber-to-fiber adhesion of the overall printed structure. As such these conformal coatings can be advantageously used to impart stability and / or impart anti-FBR properties to a tissue fiber structure. The frame and corresponding attached lattice structure can be transported to perform post-printing processing steps, e.g. one or more coatings, while the lattice structure remains attached to the frame. In this way, the fabrication platform can be advantageously used during printing, patterning and / or post-printing processing of the devices of the present disclosure.

[0080] In embodiments, the infill density may be a function of a number of posts on the fabrication platform and their relationship to one another (e.g., distance from adjacent posts and / or number of posts on opposing and / or adjacent sides of the frame).Material Strengths

[0081] In embodiments, the solid core, the at least one shell, and the one or more coating(s) may comprise the same or different material strengths. For example and without limitation, for a multilayer lattice structure that is comprised of a solid core, at least one shell, and at least one coating, the core may have a first material strength, the at least one shell may have a second material strength, and the at least one coating may have a third material strength. In such an example, each of the first, second, and third material strength may be the same. In otherembodiments, the first material strength, second material strength, and third material strength may be different, or may be different than the other two. In embodiments where the continuous fiber comprises two or more shells, each shell may comprise a different material strength. Additionally or alternatively, in some embodiments where a lattice structure is comprised of at least two coatings, each coating may comprise a different material strength. In an exemplary embodiment, a multilayer lattice structure is comprised of a solid core, a shell, and a coating, wherein a material strength of the shell is greater than a material strength of the core, and where a material strength of the coating is less than the material strengths of both the shell and core.Segmentation / Compartmentalization

[0082] In embodiments, a multilayer lattice structure made by the methods herein disclosed can be segmented / compartmentalized along at least a portion of a length of the continuously bioprinted fiber that forms the lattice structure. Details regarding the production of segmented / compartmentalized fiber structures is described in WO 2022 / 246550, the disclosure of which is expressly incorporated by reference herein in its entirety.

[0083] In embodiments, the core can be segmented / compartmentalized along at least a portion of the continuously bioprinted fiber that forms the lattice structure. In embodiments, at least one shell can be segmented / compartmentalized along at least a portion of the continuously bioprinted fiber that forms the lattice structure. In embodiments, both the core and the at least one shell may be segmented / compartmentalized along at least a portion of the continuously bioprinted fiber that forms lattice structure. In embodiments, one or more segments / compartments of the core and / or shell(s) may comprise dissociated cells). It may be understood that compartments may comprise the same or different materials (e.g., different hydrogel material). Furthermore, a shell can additionally or alternatively be segmented / compartmentalized, in similar fashion, depending on the desired application.

[0084] Compartment sizing may be a function of one or more variables, including but not limited to fiber size (e.g., length and / or diameter), number of shell(s) and / or coating(s), type of materials used in the process of continuous fiber generation, coating composition, and the like. In some embodiments, a fiber may be comprised of at least two segments / compartments which include dissociated cells, where other segments flanking the at least two segments / compartments are free of dissociated cells. For example and without limitation, at least two segments comprisingdissociated cells may be included in the core of a continuously bioprinted fiber of the present disclosure. In another example, at least two segments comprising dissociated cells may be included in the at least one shell. In embodiments, segment(s) comprising dissociated cells may be of greater, equal, or lesser length(s) than segment(s) lacking the cells. In some embodiments, spacing between compartments / segments inclusive of biological material (e.g., cells) in a fiber of the present disclosure may be between 1-5 mm, for example 1 mm, 2 mm, 3 mm, 4 mm or 5 mm apart.

[0085] Other design considerations for compartmentalization can, by example, include optimizing oxygen and nutrient diffusion for improved viability and function as well as switching core and / or shell of a fiber structure between different materials, cell types and densities. Compartmentalization can also permit printing of a fiber structure with a different therapeutic dose without changing the geometry of the fiber structure.

[0086] Segments / compartments may comprise biological materials, for example, dissociated cells of particular densities. In embodiments, the density may be the same or different between compartments. In embodiments, the biological material in compartments may be the same, or may be different. In embodiments, density of the biological material may be selected as a function of one or more of particular application (e.g., treatment of particular disease / condition), cell viability determinants, material (e.g., biocompatible material) in which the biological material is included, and the like.

[0087] In embodiments, one or more segments / compartments comprising biological material may be flanked by segments that comprise, for example, materials with immunoprotective properties. For illustrative purposes and without limitation, an immunoprotective hydrogel material may comprise, for example, a functionalized alginate including but not limited to methacrylated alginate, alginate furan, alginate thiol, alginate maleimide, and covalent click alginates (e.g., alginate blended with DMAPS-Alg and / or DMAPS-Hzd). For example, in a case in which the core of a lattice structure as herein disclosed includes one or more segments comprising biological material, the one or more segments may be flanked by other segments that comprise immunoprotective materials as herein disclosed. In another example, in a case in which a shell of a lattice structure as herein disclosed includes one or more segments comprising biological material, the one or more segments may be flanked by other segments that compriseimmunoprotective materials as herein disclosed. It is also within the scope of this disclosure that where a core of a bioprinted fiber comprises biological material, the at least one shell and / or at least one coating can comprise materials with immunoprotective properties. In another embodiment where a shell of a lattice structure comprises biological material, another shell and / or at least one coating can comprise materials with immunoprotective properties.Input Materials

[0088] Aspects of the invention include input materials that can be used for printing lattice structures for advantageous use as biomaterials. “Biomaterial” as used herein refers to a natural or synthetic substance that is useful for constructing or replacing tissue, e.g. human tissue with or without living cells. In the field of bioprinting, the term “biomaterial” is often synonymous with the term “bioink.” A number of such materials have been described above, with further elaboration below.

[0089] An input material will generally comprise at least one cross-linkable material, e.g., hydrogels including but not limited to, alginate (e.g., SLG-100 alginate), chitosan, PEGDA, PEGTA, Hyaluronic acid (HA), HAMA, collagen, CollMA, gelatin, gelMA, agarose, gellan, fibrin (fibrinogen), PVA, and the like, or any combination thereof, as well as non-hydrogels including but not limited to, PCL, PLGA, PLA, and the like, or any combination thereof. In preferred embodiments an input material comprises at least one hydrogel. Non-limiting examples of hydrogels include alginate, agarose, collagen, fibrinogen, gelatin, chitosan, hyaluronic acid-based gels, or any combination thereof. A variety of synthetic hydrogels are known and can be used in embodiments of the systems and methods provided herein. For example, in some embodiments, one or more hydrogels form at least part of the structural basis for three-dimensional structures that are printed. In some embodiments, a hydrogel has the capacity to support growth and / or proliferation of one or more cell types, which may be dispersed within the hydrogel or added to the hydrogel after it has been printed in a three dimensional configuration.

[0090] In embodiments, a hydrogel is cross-linkable by a chemical cross-linking agent. For example, a hydrogel comprising alginate may be cross-linkable in the presence of a divalent cation such as calcium chloride (CaCh), a hydrogel containing chitosan may be cross-linked using a polyvalent anion such as sodium tripolyphosphate (STP), a hydrogel comprising fibrinogen maybe cross-linkable in the presence of an enzyme such as thrombin, and a hydrogel comprising collagen, gelatin, agarose or chitosan may be cross-linkable in the presence of heat or a basic solution.

[0091] In embodiments hydrogel fibers may be generated through a precipitation reaction achieved via solvent extraction from the input material upon exposure to a cross-linker material that is miscible with the input material. Non-limiting examples of input materials that form fibers via a precipitation reaction include collagen and polylactic acid (PLA). Non-limiting examples of cross-linking materials that enable precipitation-mediated hydrogel fiber formation include polyethylene glycol (PEG) and alginate. Cross-linking of the hydrogel will increase the hardness of the hydrogel, in some embodiments allowing formation of a solidified hydrogel.

[0092] In some embodiments, a hydrogel comprises alginate. Alginate forms solidified colloidal gels (high water content gels, or hydrogels) when contacted with divalent cations. Any suitable divalent cation can be used to form a solidified hydrogel with an input material that comprises alginate. In the alginate ion affinity series Cd2+>Ba2+>Cu2+>Ca2+>Ni2+>Co2+>Mn2+, Ca2+is the best characterized and most used to form alginate gels (Ouwerx, C. et al., Polymer Gels and Networks, 1998, 6(5):393-408). Studies indicate that Ca-alginate gels form via a cooperative binding of Ca2+ions by poly G blocks on adjacent polymer chains, the so-called “egg-box” model (ISP Alginates, Section 3 : Algin-Manufacture and Structure, in Alginates: Products for Scientific Water Control, 2000, International Specialty Products: San Diego, pp. 4-7). G-rich alginates tend to form thermally stable, strong yet brittle Ca-gels, while M-rich alginates tend to form less thermally stable, weaker but more elastic gels. In some embodiments, a hydrogel comprises a depolymerized alginate.

[0093] In some embodiments, a hydrogel is cross-linkable using a free-radical polymerization reaction to generate covalent bonds between molecules. Free radicals can be generated by exposing a photoinitiator to light (often ultraviolet), or by exposing the hydrogel precursor to a chemical source of free radicals such as ammonium peroxodi sulfate (APS) or potassium peroxodi sulfate (KPS) in combination with N,N,N,N-Tetramethylethylenediamine (TEMED) as the initiator and catalyst respectively. Non-limiting examples of photo cross-linkable hydrogels include: methacrylated hydrogels, such as hyaluronic acid methacrylate (HAMA), gelatin methacrylate (GEL-MA) or polyethylene (glycol) acrylate-based (PEG-Acylate) hydrogels, which are used incell biology due to their inertness to cells. Polyethylene glycol diacrylate (PEG-DA) is commonly used as scaffold in tissue engineering, since polymerization occurs rapidly at room temperature and requires low energy input, has high water content, is elastic, and can be customized to include a variety of biological molecules.

[0094] In embodiments, an input material comprises a non-biodegradable polymer. In examples the input material may be a synthetic polymer, for example polyvinyl acetate (PVA). In embodiments, an input material may comprise hyaluronic acid (HA).

[0095] In some embodiments, a hydrogel comprises a chemically modified alginate. In examples, the chemically modified alginate comprises alginate functionalized with methacrylate groups, referred to herein as “Alg-MA.” In some embodiments, the Alg-MA can be used in an immunoprotective shell layer via blending with zwitterionic alginate, referred to herein as “Alg- zw.” Because of a dual cross-linking capability of Alg-MA, in embodiments the Alg-MA may be first printed with Alg-zw via physical cross-linking. Upon printing the fibers can then be further irradiated to induce covalent cross-linking across fibers thus resulting in F-F adhesion. In some embodiments, the chemically modified alginate may comprise thiolated alginate.

[0096] In some embodiments, one or more synthetic components may be added into hydrogel materials. Synthetic components may be useful in increasing fiber-to-fiber adhesion and / or in vivo stability. In examples, a material may comprise an acrylated zwitterionic monomer (e.g., sulfobetaine methacrylate (SBMA) and a cross-linker (e.g., polyethylene glycol) diacrylate (PEGDA). In such an example, photomediated cross-linking of the zwitterionic monomer with PEGDA may render the resultant cross-linked polymer matrix superhydrophilic, and hence, less prone to foreign body response (see WO 2022 / 246550, the contents of which is expressly incorporated by reference herein in its entirety).

[0097] In some embodiments, hydrogel materials may be cross-linked via click chemistry. For example, copolymers comprising a zwitterionic monomer and aldehyde motifs (e.g., [2- (Methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide (DMAPS)-aldehyde, referred to herein as “DMAPS-Ald”), and zwitterionic monomer and hydrazide motifs (e.g., DMAPS-hydrazide, referred to herein as “DMAPS-Hzd”), may be used (see WO 2022 / 246550). Aldehyde reacts readily with hydrazide, forming covalently cross-linked hydrogels. Because of the presence of zwitterionic monomer in the polymer backbone, these polymers may exhibit lowprotein binding properties. In embodiments, one of these polymers may be blended with alginate in a shell. Following printing, the structure may be submersed in a solution containing the counter component that will in turn result in a covalently cross-linked bridge between fibers leading to F- F adhesion.

[0098] In embodiments, an input material comprises microparticles, “Microparticles” as used herein refers to immiscible particles in the range of about 0. lum to about lOOum that are typically composed of a polymer, a metal, or other inorganic material. They can be symmetrical (e.g. spherical, cubic, etc) although this is not a requirement. Microparticles having an aspect ratio of 2: 1 or greater may be considered a microrod or microfibre.

[0099] Input materials in accordance with embodiments herein can comprise any of a wide variety of natural or synthetic polymers that support the viability of living cells, including, e.g., alginate, laminin, fibrin, hyaluronic acid, poly(ethylene) glycol based gels, gelatin, chitosan, agarose, or combinations thereof. In some embodiments, the subject bioink compositions are physiologically compatible, z.e., conducive to cell growth, differentiation, communication, and other various cell functions (e.g., release of insulin). In certain embodiments, an input material comprises one or more physiological matrix materials, or a combination thereof. By “physiological matrix material” is meant a biological material found in a native mammalian tissue. Non-limiting examples of such physiological matrix materials include: fibronectin, thrombospondin, glycosaminoglycans (GAG) (e.g., hyaluronic acid, heparin sulfate, chondroitin- 6-sulfate, dermatan sulfate, chondroitin-4-sulfate, or keratin sulfate), deoxyribonucleic acid (DNA), adhesion glycoproteins, and collagen (e.g., collagen I, collagen II, collagen III, collagen IV, collagen V, collagen VI, or collagen XVIII). Such physiological matrix material may contribute to, e.g., pancreatic p-cell survival, proliferation, and / or insulin secretion in the context of lattice structures of the present disclosure (Riopel M, and Wang R., Frontiers in Bioscience (Landmark Ed). (2014); 19(1): 77-90); Nikolova G et al., Dev Cell. (2006); 10(3): 397-405; Johansson A et al., Diabetologia. (2009); 52(11): 2385-94).Additional Fluids

[0100] Aspects of the invention include one or more buffer solutions. Buffer solutions in accordance with embodiments of the invention are miscible with an input material (e.g., ahydrogel) and do not cross-link the input material. In some embodiments, a buffer solution comprises an aqueous solvent. Non-limiting examples of buffer solutions include polyvinyl alcohol, water, glycerol, propylene glycol, sucrose, gelatin, or any combination thereof.

[0101] Buffer solutions in accordance with embodiments of the invention can have a viscosity that ranges from about 1 mPa s to about 5,000 mPa s, such as about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 1,250, 1,500, 1,750, 2,000, 2,250, 2,500, 2,750, 3,000, 3,250, 3,500, 3,750, 4,000, 4,250, 4,500, or 4,750 mPa s. In some embodiments, the viscosity of a buffer solution can be modulated so that it matches the viscosity of one or more input materials.

[0102] Aspects of the invention include one or more sheath fluids. Sheath fluids in accordance with embodiments of the invention are fluids that can be used, at least in part, to envelope or “sheath” an input material being dispensed from a dispensing channel. In some embodiments, a sheath fluid comprises an aqueous solvent. Non-limiting examples of sheath fluids include polyvinyl alcohol, water, glycerol, propylene glycol, sucrose, gelatin, or any combination thereof. Sheath fluids in accordance with embodiments of the invention can have a viscosity that ranges from about 1 mPa s to about 5,000 mPa s, such as about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 1,250, 1,500, 1,750, 2,000, 2,250, 2,500, 2,750, 3,000, 3,250, 3,500, 3,750, 4,000, 4,250, 4,500, or 4,750 mPa s. In some embodiments, the viscosity of a sheath fluid can be modulated so that it matches the viscosity of one or more input materials.

[0103] In some embodiments, a sheath fluid comprises a chemical cross-linking agent. In some embodiments, a chemical cross-linking agent comprises a divalent cation. Non-limiting examples of divalent cations include Cd2+, Ba2+, Cu2+, Ca2+, Ni2+, Co2+, or Mn2+. In a preferred embodiment, Ca2+is used as the divalent cation. In some embodiments, the concentration of a divalent cation in the sheath fluid ranges from about 80 mM to about 140 mM, such as about 90, 100, 110, 120 or 130 mM.Cell Populations

[0104] In embodiments, the cell population is selected from the group comprising or consisting of a single-cell suspension, cell aggregates, cell spheroids, cell organoids, or combinations thereof. Input materials in accordance with embodiments of the invention can incorporate any mammalian cell type, including but not limited to stem cells (e.g., embryonic stem cells, adult stem cells, induced pluripotent stem cells), germ cells, endoderm cells (e.g., lung, liver, pancreas, gastrointestinal tract, or urogenital tract cells), mesoderm cells (e.g., kidney, bone, muscle, endothelial, or heart cells), ectoderm cells (skin, nervous system, pituitary, or eye cells), stem cell- derived cells, or any combination thereof. In preferred embodiments, at least one cell population comprises or consists of hepatocytes.

[0105] Cells can be obtained from donors (allogenic), from a different species to the recipient (xenogeneic), or from recipients (autologous). Specifically, in embodiments, cells can be obtained from a suitable donor, such as a human or animal, or from the subject into which the cells are to be implanted. Mammalian species include, but are not limited to, humans, monkeys, dogs, cows, horses, pigs, sheep, goats, cats, mice, rabbits, and rats. In one embodiment, the cells are human cells. In other embodiments, the cells can be derived from animals such as dogs, cats, horses, monkeys, or any other mammal.

[0106] In embodiments, the cell population comprises primary cells from endocrine and exocrine glands selected from the group consisting of pancreas, liver, kidney, parathyroid, thyroid, pineal gland, pituitary gland, thymus, adrenal gland, ovary, testis, tonsillar, enteroendocrine cells, stem cells, stem-cell derived cells of any of the foregoing primary cell types, or cells engineered to secrete a therapeutic agent. In embodiments, the cell population comprises HepG2s, primary human hepatocytes (PHHs), mesenchymal stem cells (MSCs), embryonic stem cell- or induced pluripotent stem cell-derived hepatocyte-like cells, liver stem cell-derived hepatocyte-like cells, xenogeneic hepatocytes, genetically engineered stem cell-derived hepatocytes, stem-derived or primary endothelial cells, and combinations thereof. In some embodiments, the dissociated cells comprise adrenal cells. In some embodiments, the adrenal cells can be selected from the group consisting of primary adrenal cells, including primary adrenal cortical cells derived from any cell layer of the adrenal cortex (zona glomerulosa, fasciculata and / or reticularis), primary adrenal medullary cells, embryonic stem cell- or induced pluripotent stem-cell derived adrenal cortical- like cells, adrenal stem cell-derived adrenal cortical-like cells, genetically engineered stem cell- derived adrenal cortical cells, NCI-H295R cells and combinations thereof.

[0107] Because hepatocytes within the solid fiber herein disclosed are protected from the host immune system, hepatocytes can be derived from any suitable source, i.e., human or non-human. In embodiments, the hepatocytes are stem or progenitor cells, including induced pluripotent stem cells that differentiate into hepatocytes. Suitable hepatocyte populations (including but not limited to mature hepatocyte, immature hepatocyte, fetal like hepatocyte, hepatocyte-like cells, hepatic progenitor cells, liver precursor cells) and methods for producing such populations are known in the art, see e.g., Patents, patent publications, or patent applications US20210189350A1, US20180030415A1, US8148151B2, US10683486B2, US20200308551A1, US20200157494A1, US20220233605A1, US9284576B2, US10227565B2, US10004826B2, US8569573B2, US9284531B2, WO2020227711A1, US20190084944A1, US10081609B2, US20170191031A1, US7989204B2, US8574905B2. Publications Si-Tayeb et al., (2010) Hepatology; Hannan, et al., (2013) Nature protocols; Ogawa et al., (2013) Development; Magner et al. (2013) Stem cells; Zhu et al., (2014) Nature; Siller et al., (2015) Stem Cell Reports; Blackford et al., (2019) Stem cells translational medicine; Raggi et al., (2022) Stem Cell Reports; Blackford et al., (2023). Biomaterials; Carpentier et. al., (2014). The Journal of clinical investigation; Carpentier, et al., (2016). Stem cell research; Xia et al., (2017). Journal of hepatology; Uchida et. al., (2021). Molecular therapy; Tilson et. al., (2021). Hepatology; Duan et al., (2007). Stem cells; Bandi et al., (2019). Differentiation; Mallanna and Duncan (2013). Curr Protoc Stem Cell Biol., each of which are hereby incorporated by reference in their entirety. Stem cell derived hepatocytes or kits to differentiate stems cells into hepatocytes are commercially available from suppliers including but not limited to Creative Bioarray (Human iPSC-derived Hepatocytes, Cat.No. CSC-00863L), STEMCELL Technologies (STEMdift™ Hepatocyte Kit, Catalog # 100-0520).

[0108] In embodiments, the hepatocytes are primary hepatocytes from any suitable source, i.e., human or non-human. Primary hepatocytes are commercially available from suppliers including but not limited to Thermo Fisher human hepatocytes (Cat# HMCPSQ and HMCPTS), KaLy Cell (Plateable CryoHeps), Lonza (PHH cells Cat# HUCP), and AnaBio (Primary Human Hepatocytes). In embodiments, the hepatocytes can be encapsulated with other cell types, including but not limited to Mesenchymal stem cells (MSCs). Mesenchymal stem cells (MSCs) are commercially available from suppliers including but not limited to RoosterBio (Xeno-Free RoosterVial™-hUC, Cat# C43002UC or C43001UC and RoosterVial™-hUC-20M-CC" Cat# C04020UC).

[0109] In some embodiments, the dissociated cells comprise HepG2s, primary human hepatocytes (PHHs), mesenchymal stem cells (MSCs), embryonic stem cell- or induced pluripotent stem cell-derived hepatocyte-like cells, liver stem cell-derived hepatocyte-like cells, xenogeneic hepatocytes, genetically engineered stem cell-derived hepatocytes, stem-derived or primary endothelial cells, and combinations thereof. In some embodiments, the dissociated cells comprise HepG2s, primary human hepatocytes (PHHs), and / or mesenchymal stem cells (MSCs), and combinations thereof.

[0110] In some embodiments, the dissociated cells comprise a 3: 1 ratio of PHHs and MSCs.

[0111] In some embodiments, the at least one biological material included in a lattice structure of the present disclosure comprises a cell population expressing / secreting one or more endogenous biologically active agent(s), e.g., insulin, glucagon, ghrelin, pancreatic polypeptide, Factor VII, Factor VIII, Factor IX, alpha- 1 -antitrypsin, an angiogenic factor, a growth factor, a hormone, an antibody, an enzyme, a protein, an exosome, and the like. Discussed herein, endogenous biologically active agents comprise those agents that the cell naturally produces in a biological context (e.g., insulin release in response to elevated glucose concentrations). An endogenous biologically active agent can constitute a therapeutic agent in the context of the present disclosure.

[0112] In some embodiments, an input material can comprise genetically engineered cells that secrete specific factors. It is within the scope of this disclosure that a cell population as discussed above can comprise, in embodiments, engineered cells (e.g., genetically engineered cells) that secrete specific factors. Cells can also be from established cell culture lines, or can be cells that have undergone genetic engineering and / or manipulation to achieve a desired genotype or phenotype. In some embodiments, pieces of tissue can also be used, which may provide a number of different cell types within the same structure.

[0113] Genetic engineering techniques applicable to the present disclosure can include but are not limited to recombinant DNA (rDNA) technology (Stryjewska et al., Pharmacologial Reports. 2013; 65: 1075), cell-engineering based on use of targeted nucleases (e.g., meganuclease, zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN), clustered regularly interspaced short palindromic repeat-associated nuclease Cas9 (CRISPR-Cas9), etc. (Lim et al., Nature Communications. 2020; 11 : 4043; Stoddard BL, Structure. 2011; 19(1): 7-15; Gaj et al., Trends Biotechnol. 2013; 31(7): 397-405; Hsu et al., Cell. 2014; 157(6): 1262; Miller et al.,Nat Biotechnol. 2010; 29(2): 143-148), cell-engineering based on use of site-specific recombination using recombinase systems (e.g., Cre-Lox) (Osborn et al., Mol Ther. 2013; 21(6): 1151-1159; Hockemeyer et al., Nat Biotechnol. 2009; 27(9): 851-857; Uhde-Stone et al., RNA. 2014; 20(6): 948-955; Ho et al., Nucleic Acids Res. 2015; 43(3): el7; Sengupta et al., Journal of Biological Engineering. 2017; 11(45): 1-9), and the like. In some embodiments, some combination of the above-mentioned techniques for cell-engineering may be used.[00114J Encompassed by the present disclosure are engineered cells capable of producing one or more therapeutic agents, including but not limited to proteins, peptides, nucleic acids (e.g., DNA, RNA, mRNA, siRNA, miRNA, nucleic acid analogs), peptide nucleic acids, aptamers, antibodies or fragments or portions thereof, antigens or epitopes, hormones, hormone antagonists, growth factors or recombinant growth factors and fragments and variants thereof, cytokines, enzymes, antibiotics or antimicrobial compounds, anti-inflammation agent, antifungals, antivirals, toxins, prodrugs, small molecules, drugs (e.g., drugs, dyes, amino acids, vitamins, antioxidants) or any combination thereof.

[0115] In embodiments, cells of the present disclosure may be modified to comprise at least one mechanism for providing a local immunosuppression at a transplant site when transplanted in an allogeneic host, for example in tissue fibers of the present disclosure. In examples, a cell or cells may comprise a set of transgenes, each transgene encoding a gene product that is cytoplasmic, membrane bound, or local acting, and whose function can include but is not limited to mitigate antigen presenting cell activation and function, to mitigate graft attacking leukocyte activity or cytolytic function; to mitigate macrophage cytolytic function and phagocytosis of allograft cells; to induce apoptosis in graft attacking leukocytes; to mitigate local inflammatory proteins; and to protect against leukocyte-mediated apoptosis (WO2018 / 227286; Harding et al., BioRxiv. 2019; DOI: 10.1101 / 716571; Lanza et al., Nature Reviews Immunology. 2019; 19: 723-7331; Harding et al., Cell Stem Cell. 2020; 27(2): 198-199).

[0116] In embodiments, cells of the present disclosure may be modified in a manner to exert control over cell proliferation. As an example, a cell may be genetically modified at a cell division locus (CDL) to comprise a negative selectable marker and / or an inducible activator-based gene expression system, thereby enabling control over the permitting, ablation and / or inhibition ofproliferation of the genetically modified cells by addition or removal of an appropriate inducer (W02016 / 141480; Liang etal., Nature. 2018; 563(7733): 701-704).

[0117] Appropriate growth conditions for mammalian cells are well known in the art (Freshney, R. I. (2000) Culture of Animal Cells, a Manual of Basic Technique. Hoboken N. J., John Wiley & Sons; Lanza et al. Principles of Tissue Engineering, Academic Press; 2nd edition May 15, 2000; and Lanza & Atala, Methods of Tissue Engineering Academic Press; 1 st edition October 2001). Cell culture media generally include essential nutrients and, optionally, additional elements such as growth factors, salts, minerals, vitamins, etc., that may be selected according to the cell type(s) being cultured. Particular ingredients may be selected to enhance cell growth, differentiation, secretion of specific proteins, etc. In general, standard growth media include Dulbecco's Modified Eagle Medium, low glucose (DMEM), with 110 mg / L pyruvate and glutamine, supplemented with 10-20% fetal bovine serum (FBS) or calf serum and 100 U / ml penicillin are appropriate as are various other standard media well known to those in the art. Growth conditions will vary depending on the type of mammalian cells in use and the tissue desired.

[0118] In some embodiments, cell-type specific reagents can be advantageously employed in the subject input materials for use with a corresponding cell type. For example, an ECM can be extracted directly from a tissue of interest and then solubilized and incorporated it into an input material to generate tissue- specific input materials for printed tissues. Such ECMs can be readily obtained from patient samples and / or are available commercially from suppliers such as zPredicta (rBone™, available at zpredicta.com / home / products).Printing Systems

[0119] In preferred embodiments, bioprinting systems comprise technology as described in WO20 14 / 197999, WO2018 / 165761, W02020 / 056517, WO2021 / 081672, WO2022 / 246550 and WO2023 / 108302, the disclosures of which are expressly incorporated herein by reference. As detailed therein, the disclosed bioprinting systems and components thereof enable multi-material switching, and hence the composition of one or more components (e.g., cell type, biomaterial composition) of the synthetically generated tissue fiber can be modified along the length of the fiber while continuously printing. In embodiments, a microfluidics-based bioprinting system isthe RX1™ bioprinter (Aspect Biosystems, Vancouver, BC, Canada), or the RX2™ bioprinter system described in WO 2023 / 108302, the contents of which is incorporated by reference herein in its entirety.

[0120] In an exemplary embodiment of a preferred bioprinting system, the system comprises a print head comprising a dispensing channel, wherein one or more material channels and a core channel converge at the proximal end of the dispensing channel. A print head may be configured to dispense buffer solution and / or sheath fluid simultaneous with one or more cross-linkable materials. In some embodiments, a print head is configured to maintain a constant mass flow rate through the dispensing channel. In this manner, a print head can be configured to facilitate a smooth and continuous flow of one or more input materials (or a mixture of one or more input materials) and a buffer solution and / or sheath fluid through the dispensing channel. In use of such a print head, an input material flowing through the dispensing channel can be cross-linked from the inside, by a fluid flowing through the core channel and / or from the outside, by sheath fluid flowing through a downstream sheath fluid channel, as described more particularly in W02020 / 056517. In some embodiments, a print head comprises one or more fluidic focusing chambers comprised of a conical frustum shape and, optionally, one or more print head adaptors, as described in detail in WO 2021 / 081672, WO 2022 / 246550 and WO 2023 / 108302. In embodiments, a print head is the DUO™ microfluidic printhead, or the CENTRA™ microfluidic printhead (Aspect Biosystems, Vancouver, BC, Canada).

[0121] Other examples of bioprinting systems relevant in the context of the present disclosure, for example those that can be modified or used in conjunction with the methods of the present disclosure, include but are not limited to 3-D Bioplotter® (EnvisionTEC Inc., Dearborn, MI, USA), NovoGen Bioprinter® Platform (Organovo®, San Diego, CA, USA), R-Gen 100 and R- Gen 200 (RegenHU, Villas-Saint-Pierre, Switzerland), Bioprinter Fabion and Fabion 2 (3D Bioprinting Solutions, Moscow, Russia), BioBot® Basic, BioAssemblyBot® 200 / 400 / 500 (Advanced Solutions, Louisville, KY, USA), BIO X™, BIO X6™, INKREDIBLE+™ (CellINK, Boston, MA, USA), Ourobotics Revolution (Ourobotics, Cork, Ireland), BioScaffolder 2.1 (GeSim, Radeberg, Germany), Omega Bioprinter (3Dynamic Systems, Bridgend, UK), SynAand Explorer (Bio3D, Singapore), Alevi 1 / 2 / 3 (Alevi by 3D Systems, Rock Hill, SC, USA), and Dr. Invivo 4D6 (Rokit Healthcare, Seoul, South Korea).Methods of Making

[0122] Aspects of the invention include a) providing a bioprinting system comprising a print head comprising a plurality of microfluidic channels to selectively provide a core material, at least one shell material, and a sheath fluid to a dispensing orifice; a receiving surface for receiving the materials dispensed from the orifice, a positioning unit for positioning the orifice of the print head in three dimensional space, the positioning unit operably coupled to the print head, and at least one dispensing means for dispensing the fiber from the dispensing orifice; b) via the bioprinting system, simultaneously printing the core and the at least one shell of the tissue fiber, wherein the cell population is encapsulated in the core and / or in the at least one shell; and c) cryopreserving said bioprinted tissue fiber with a cryoprotectant; wherein the cell population comprises at least 95% dissociated cells embedded in a biocompatible matrix, and wherein the fiber is cryopreserved prior to aggregation of the dissociated cells; preferably with 15 minutes to 3 hours of bioprinting, more preferably with 30, 45 or 60 minutes of bioprinting.

[0123] In embodiments, the cryopreserving step comprises adding cryoprotectant to the printed fiber atleast 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 minutes before freezing. In embodiments, the core material further comprises a viscosifier, preferably wherein the viscosity of the core material and the shell material are substantially the same.

[0124] In some embodiments, the cryoprotectant does not include dimethyl sulfoxide (DMSO). In some embodiments, a cryoprotectant can include DMSO. In some embodiments, the DMSO-containing cryoprotectant is selected from the group consisting of CRYOSTOR(R) CS10, CRYOSTOR(R) CS5, CROSTOR CS2, PSC Cryopreservation Kit, Synth-a-Freeze™ Cry opreservation Medium, CELLBANKER(R) 1, CELLBANKER(R) 2, and STEM- CELLBANKER(R) GMP Grade. In some embodiments, the DMSO-free cryoprotect is selected from the group consisting of CRYOSTOR(R) CSB, CrySOfree™ DMSO-free Cry opreservation Medium, STEM-CELLBANKER(R) DMSO-free, GMP Grade, Cell-Vive™ CD DMSO-Free Cryopreservation Solution.

[0125] In embodiments, the printing step may comprise dispensing the continuously bioprinted core / shell fiber on the receiving surface to form a multilayer (e.g. lattice) structure, and wherein the multilayer structure is cryopreserved prior to aggregation of the dissociated cells inthe fiber. In embodiments, the receiving surface may comprise a fabrication platform for supporting the continuously bioprinted core / shell fiber, the fabrication platform comprising a frame defining a void and comprising a plurality of posts on opposing sides of the frame for securing and suspending the continuously bioprinted fiber during printing; and the printing step comprises dispensing the fiber around a plurality of said posts to form a lattice structure comprising at least two, three, four, or five layers of the fiber. In embodiments, the method can further comprise coating the multilayer (e.g. lattice) structure after printing is completed.

[0126] The general manner of printing a first layer and, optionally a second layer, third layer, and so on, is described in detail above and, for example, in International Patent Application No. PCT / CA2023 / 050668 and WO 2022 / 246550, each of which is incorporated herein by reference in its entirety. Broadly speaking, the process flow includes generating a multilayer structure from a continuous fiber, preferably via a fabrication platform, and optionally applying at least one coating to obtain the implantable device. The continuous fiber is generally produced in a manner whereby the continuous fiber is exposed to a sheath fluid containing cross-linker during printing that crosslinks the continuous fiber from the outside in, but in other embodiments inside-out cross-linking where cross-linker is included as part of a core material is also within the scope of this disclosure (see WO 2022 / 246550). It should also be understood that fibers comprising more than one shell are within the scope of this disclosure.

[0127] As the fiber is printed, the sheath fluid is removed, for example via flowing through a porous receiving surface. Following bioprinting, the method optionally further comprises submerging the multilayer structure in cross-linking solution or otherwise applying cross-linking solution, for example via spraying or dispensing via the same dispensing means used to dispense the continuous fiber) to facilitate / continue uniform cross-linking of the entire printed lattice structure. A coating can also be applied, for example, by submerging structure into a coating solution, by using a microfluidic print head (e.g., the same microfluidic print head used to fabricate the fiber) to dispense coating solution onto the completed structure, spraying the structure with coating solution, and the like.

[0128] In embodiments, the coating solution comprises a cross-linkable material (e.g., alginate). In embodiments, the coating solution comprises a material that is the same as the material comprising the core and / or at least one shell of the structure being coated. In otherembodiments, the coating solution may comprise a material that is different than the material comprising the core and / or at least one shell. In embodiments, residual cross-linker (e.g., Ca2+) associated with the structure contributes to initial cross-linking of the material in the coating solution with the material comprising the shell. Following coating of the lattice structure, the structure can be submerged in cross-linking solution, or otherwise applying cross-linking solution, for example via spraying or dispensing via the same microfluidic print head used to dispense the continuous fiber. It is within the scope of this disclosure that different cross-linking solutions can be used, for example in a case where the material used to fabricate the at least one shell is comprised of a different material than the material used to fabricate a coating. In this way, the coating can be added uniformly to an entirety of the multilayered structure produced from a continuously bioprinted fiber. Although not specifically illustrated, it is within the scope of this disclosure that a second coating can be further applied in similar fashion as that of the first coating.

[0129] In embodiments where a single coating is applied, the coating may impart stability and / or fiber-to-fiber adhesion properties and / or anti-FBR properties to the resultant fiber structure. In embodiments where a fiber structure comprises two coatings, a first coating may have particular desired properties (e.g., materials selected for enhancing stability and / or F-F adhesion) while the second coating may have additional / alternative properties (e.g., tailored to have anti-FBR properties. In preferred embodiments, the coating is a conformal coating that uniformly covers the entirety of the exposed surfaces of the fiber structure.

[0130] In embodiments, a coating is comprised of a hydrogel material, for example a hydrogel material comprising one or more of alginate, zwitterionic alginate, SBMA, chitosan, PEGDA, PCL, PEG, poly-L-lysine (PLL), PEGTA, Hyaluronic acid (HA), HAMA, collagen, CollMA, gelatin, gelMA, agarose, gellan, fibrin (fibrinogen), PVA, and the like, or any combination thereof. In some examples, the coating is comprised of a functionalized alginate, i.e., an alginate that is chemically modified to include one or more properties, including but not limited to immunoprotective properties that are advantageous in the manufacture of lattice structures of the present disclosure. Examples of functionalized alginates include but are not limited to methacrylated alginate, alginate furan, alginate thiol, alginate maleimide, and covalent click alginates (e.g,, alginate blended with DMAPS-Alg and / or DMAPS-Hzd).

[0131] In embodiments, a coating is of a material strength that is less than the material strength of one or more shell(s) and / or core. In embodiments where the structure comprises two coatings, an outer coating may be of a material strength that is lesser than a material strength of an inner coating, or vice versa. Alternatively, the outer coating and inner coating may be of substantially the same material strength, optionally where the outer coating and inner coating are comprised of different materials. In embodiments, the core is solid, optionally wherein the core is segmented / compartmentalized along the length of the fiber. In additional or alternative embodiments at least one shell may be segmented / compartmentalized along the length of the fiber.

[0132] As noted, the conformal coating may impart stability to the fiber structure, and / or may endow the structure with properties that optimize the interface between the fiber structure and host, e.g. anti-FBR properties, promotion of vascularization, etc. Further details on the production of such conformal coatings is described in International Patent Application No. PCT / CA2023 / 050667, published as WO 2023 / 220816, the contents of which is expressly incorporated by reference herein in its entirety.

[0133] In embodiments, a bioprinted fiber structure made by the methods herein disclosed can also be segmented / compartmentalized along at least a portion of a length of a fiber, preferably a continuous fiber, that makes up the bioprinted fiber structure. Details of the production of segmented / compartmentalized bioprinted fiber structures is described in WO 2022 / 246550, the contents of which is expressly incorporated by reference herein in its entirety.

[0134] In some embodiments, a method first comprises providing a design for a multilayer structure as herein disclosed to be printed. The design can be created using commercially available CAD software. In some embodiments, the design comprises information regarding specific materials (e.g., for heterogeneous structures comprising multiple materials) to be assigned to specific locations in the structure(s) to be printed.Methods of Treatment

[0135] The bioprinted 3D lattice structures of the present disclosure are useful in methods of treating a disease or condition in a subject in need thereof. Aspects of the invention include a method of implanting a cell population into a subject in need thereof, comprising: a. thawing acryopreserved tissue fiber or composition / medical device of the present disclosure; and b. implanting said fiber or said composition / medical device into said subject. In embodiments, the method can further include culturing said tissue fiber or said composition / medical device in fresh culture media after thawing until the dissociated cells form aggregates in said tissue fiber, prior to implantation into said subject. In embodiments, thawing is performed using a suitable thawing device, e.g., a ThawSTAR® CFT2, ThawSTAR CB, or the like[00136J In embodiments, the disease or condition is a disease or condition of the liver.

[0137] The methods involve thawing a cryopreserved tissue fiber or composition / medical device as herein described comprising a therapeutically effective amount of dissociated cells (e.g., hepatocytes) encapsulated within the fiber and / or structure and, optionally, culturing said tissue fiber or said composition / medical device in fresh culture media after thawing until the dissociated cells form aggregates in said tissue fiber, prior to implantation into said subject. The therapeutically effective amount of cells may comprise some predetermined therapeutically effective number cells. Therapeutically effective amounts can also vary according to, for example, the size and health of the individual being treated. For example, a therapeutically effective amount can comprise some number / amount of hepatocytes per kilogram of body weight.

[0138] The methods involve implanting a fiber and / or structure as herein disclosed into a subject in need thereof. In embodiments the methods comprise implanting one or more times. For example, in embodiments the methods comprise implanting into a subject in need thereof a thawed fiber or composition / medical device comprising a therapeutically effective amount of cells about once every 4 months, once every 6 months, once a year, once every two years, once every three years, once every four years, once every five years, or more. In embodiments, the encapsulated cell population encapsulated survives for at least for at least four months, or at least 6 months, or at least one year, or at least two years, or at least three years, or at least four years, or at least five years, or more, following implantation. In embodiments, a subject may require just a single implant. In embodiments, the composition / medical device may need to be replaced once every 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 months, once every 1, 2, 3, 4, 5 or more years.

[0139] In embodiments, the composition / medical device comprising the cell population of interest is implanted to the greater omentum of the subject. The greater omentum (also known as the great omentum, omentum majus, gastrocolic omentum, epiploon, or, caul) is a large apron-likefold of visceral peritoneum that hangs down from the stomach and extends from the greater curvature of the stomach back to ascend to the transverse colon before reaching to the posterior abdominal wall. Thus, the structure may be implanted into a pouch formed surgically from the omentum.

[0140] In embodiments, the composition / medical device can be surgically implanted using minimally invasive surgical techniques such as laparoscopy. In embodiments, the composition / medical device is implanted laparoscopically into the abdominal cavity or thoracic cavity. In embodiments, the implanting is carried out intraperitoneally, percutaneously, or subcutaneously.

[0141] In embodiments, the composition / medical device is anchored or immobilized (e.g., by suture) at the implantation site to maintain the structure near the implantation site. In embodiments, the delivery of the therapeutic agent is not location dependent and biodistribution of the agent is dependent on the subject’s vasculature or body fluids. In embodiments, the structure is implanted percutaneously or subcutaneously under the skin of the abdomen, forearm, flank, back, buttocks, leg, and the like, where it substantially remains until such time as it is required to be removed.

[0142] In embodiments, the composition / medical device is retrievable after implantation. In embodiments, anchoring or immobilizing the structure prevents it from migrating, moving, or traversing inside the subject, and facilitates retrieval. However, it is also within the scope of this disclosure that given the dimensions of the composition / medical device, anchoring or otherwise immobilizing it may not be needed, where retrieval remains easily facilitated. Retrieval may be desirable after the encapsulated cell population ceases or substantially ceases to function at a certain threshold, after cell death exceeds or is expected to exceed some threshold, and the like. Following retrieval, the structure can be replaced by a new one to maintain the desired effect.

[0143] It is within the scope of this disclosure that the implanted lattice structure may be monitored regularly (e.g., weekly, bi-monthly, monthly) to ensure the cells of the lattice structure are functioning as desired. For example, the structure may comprise one or more contrast agents to facilitate in vivo monitoring of fiber device placement, location of implant at some time-point after implantation, health of the implant, deleterious effects of non-target cell types, inflammation, and / or fibrosis. Suitable contrast agents include, without limitation, nanoparticles, nanocrystals,gadolinium, iron oxide, iron platinum, manganese, iodine, barium, microbubbles, fluorescent dyes, and other known to those of skill in the art.

[0144] Methods of in vivo monitoring include but are not limited to confocal microscopy, 2- photon microscopy, high frequency ultrasound, optical coherence tomography (OCT), photoacoustic tomography (PAT), computed tomography (CT), magnetic resonance imaging (MRI), single photon emission computed tomography (SPECT), and positron emission tomography (PET). These alone or combined can provide useful means to monitoring the implanted lattice structure.EXAMPLES

[0145] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the methods and compositions of the invention, and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.Example 1. Bioprinting and Assessment of Cell Populations comprising Hepatocytes and Elimination of Pre-aggregation

[0146] Solid fibers containing dissociated cells in the core of a core-shell fiber and preaggregated cell-containing fibers with the aggregated spheroids in the core of the core-shell fiber were produced using primary rat hepatocytes (PRHs).

[0147] 2.4 x 107primary rat hepatocytes were thawed, resuspended in Hepatocyte ThawMedium (Thermo Fisher) and centrifuged. 8 x 106mesenchymal stem cells were also thawed, resuspended in Rooster MSC media (RoosterBio) and centrifuged. Thawing was performed using a ThawSTAR CFR2 (Biolife Solutions). Media was decanted and cells were resuspended in 1 mL of media, combined together and re-centrifuged. Cells were then resuspended in gelatinby adding 1 part of gelatin (20% w / w stock solution) to 3 parts cells volume. Cells were then loaded in a syringe for bioprinting.

[0148] A syringe containing 2% SLF-100 alginate (Novamatrix) dissolved in saline was loaded into the shell channel of the printhead, a syringe with crosslinker solution containing 95 mM CaCh, 5 mM BaCh and 3% PVA (90 kDa) was connected to the crosslinker channel of the printhead, a bottle of 0.9% normal saline was connected to the buffer channel of the printhead. A fiber containing 1 million cells was printed and repeated 24 times.

[0149] For pre-aggregated cell bioprinting, cells were thawed as above and then used to form aggregates with an average of 199 hepatocytes per aggregate. Aggregation was performed using STEMCELL Technologies AggreWell Plates according to the manufacturer’s instructions. Cells were then cultures in William’s E media supplemented with FBS, ITS and dexamethasone.Spheroids were harvested after 3 days and centrifuged. The pellet was resuspended in 3% SLG- 100 alginate and diluted to 1.5% inclusive of ell volume.

[0150] Solid fibers (containing dissociated cells) were cultured in William’s E media with FBS, ITS and dexamethasone and then switched to media without FBS. Pre-aggregated fibers were cultured in media without FBS. Media was changed every 2 days and collected on days 1, 3, 5 and 7 to quantify albumin. Cyp3A4 activity was analyzed using a P450-Glo Cyp3A4 kit (Promega) according to the manufacturer’s instructions. Secreted albumin from conditioned media was quantified by ELISA (Abeam catalog # ab235642 or ab 179887) according to the manufacturer’s instructions.

[0151] Histology was performed 7 days post-printing. Fibers were fixed in 10% formalin for 16 hours followed by rinsing with 70% ethanol. Fibers were embedded in paraffin, sectioned and stained for H&E using standard protocols.

[0152] Live / dead assays were performed by incubation of fibers in culture media with addition of 4 uM Calcein AM, 5 ug / mL DAPI and 15 ug / mL propidium iodide for 30 minutes followed by imaging with a Zeiss AxioObserver.

[0153] For cry opreservation, within 1 hour of printing, fibers were transferred to a controlled CryoMed controlled rate freezer. The freezing protocol was to hold at 4 °C until the samples reach 5 °C, decrease the chamber temperature by 1 °C per minute until the sample reaches -4 °C,decreasing the chamber temperature by 10 °C per minute until the chamber reaches -40 °C, increasing the chamber temperature by 1 °C per minute until the chamber reaches -12 °C, decreasing the chamber temperature by 1 °C per minute until the chamber reaches -40 °C, and decreasing the chamber temperature by 10 °C per minute until the chamber temperature reaches - 90 °C. Samples were then stored in liquid nitrogen.

[0154] Vials were thawed using a 37 °C water bath until the solution was fully melted and transferred to a tissue culture dish via pouring. Cryopreservation media was then aspirated off and replaced with culture media for 1 minute, followed by aspiration of the culture media and culture according to the methods above.

[0155] For pre-aggregated fibers, there were 50 million PRH / mL, 100:50 PRH:MSC spheroids. For solid fibers, there were 75% v / v cells in the 5% fish gelatin core with a 2: 1 ratio ofPRH:MSC.

[0156] FIGS. 1A-1C show H&E staining of pre-aggregated and solid fibers fibers at day 7 post-printing (FIG. 1A), fluorescent micrographs of a live / dead assay for solid fibers and preaggregated fibers at days 1, 3 and 7 post-printing (FIG. IB) demonstrating that solid fibers had much less dead space than pre-aggregated fibers, and albumin levels for solid fibers and preaggregated fibers for two different donors (FIG. 1C) demonstrating that solid fibers had much higher albumin production. FIG. ID shows relative albumin production post-cryopreservation, demonstrating over 70% of the albumin production capacity is retained compared to noncry opreserved solid fibers, suggesting that a tissue therapeutic could be manufactured well before use in a clinical setting. FIG. IE shows albumin production for solid fibers that were cultured or which were cryopreserved and then cultured. Albumin production in cryopreserved cells was greater than 70% of non-frozen cell types, demonstrating that hepatocytes and not just MSCs survive the process. FIG. IF shows the live / dead assay for solid fibers that were or were not cryopreserved. FIG. 1G shows micrographs of solid fibers that were or were not cryopreserved and demonstrate aggregation after thawing. In addition, moderate amounts of latent heat of crystallization were observed and suggests the samples super cooled leading to some intracellular ice formation. Fibers were softer but were strong enough to be handled with forceps and did not have any obvious cracks or deformities. Cellular aggregation within solid core fibers was observed in both fresh and frozen hepatocyte fibers.

[0157] These results demonstrate that viability in solid fibers is higher when compared to pre-aggregated PRH cells, albumin production is much higher in PRH cells within solid fibers when compared to aggregates, viability is lower for PRH than historical PHH data.

[0158] The same processes were performed for primary human hepatocytes (PHHs). For pre-aggregated fibers there were 50 million PHH / mL, 100:50 PHH:MSC spheroids. For solid fibers, there were 75% v / v cells in the 5% gelatin core at a 2: 1 ratio of PHH:MSC.

[0159] FIGS. 2A-2B show fluorescent micrographs of a live / dead assay for solid fibers and pre-aggregated fibers from two different donors (FIG. 2A) and albumin and Cyp3A4 activity for solid fibers and pre-aggregated fibers from two different donors (FIG. 2B). As shown, solid fiber tissue therapeutics contain less dead space and aggregate after printing.

[0160] These results demonstrate that viability in solid fibers is higher when compared to pre-aggregated PRH cells, albumin production is much higher in PHH cells within solid fibers when compared to aggregates and Cyp3A4 activity is greatly improved with solid fiber printing.

[0161] Additionally, the solid fibers were also smaller in diameter than fibers with preaggregated cells.Example 2. Bioprinting of Tissue Therapeutics

[0162] Increasing the density of tissue therapeutics has major advantages when it comes to the overall size of the implant. Efforts were made to increase cell density of the core of tissue therapeutics to be similar to native tissue. Production of thin fibers via bioprinting can enable high density tissue therapeutic creation.

[0163] Hepatocytes were suspended in a cell-containing biomaterial and printed into a coaxial fiber with an external cell-free shell. Such fibers can optionally be patterned into three- dimensional shapes. As shown in FIG. 3 A, which shows a brightfield microscope image of a fiber and a fluorescence microscopy image of a fiber depicting cells within the fiber, the cells can be distributed in the core of the fiber. The cells can also be distributed in the core, as shown in FIG. 3B, where the cells are in the first shell (annulus).Example 3. Bioprinting and In Vivo Assessment of Tissue Therapeutics

[0164] Standard of care treatment for many liver diseases including acute liver failure (ALF) and urea cycle disorders (UCDs) consist of liver transplantation. This is effective but invasive and requires chronic immune suppression and can result in bleeding. Recent research has demonstrated promising results for encapsulated hepatocytes implanted into the intraperitoneal (IP) space as a treatment for pediatric ALF. It is unclear if this procedure can be completed at less invasive surgical sites. The subcutaneous (SubQ) space has the benefit of being comparatively non-invasive as well as being larger and in some liver diseases the IP space may be fouled with ascites. This example describes the implantation of hepatic BTTs containing primary human hepatocytes (PHHs) and mesenchymal stromal cells (MSCs) into both the IP and SubQ spaces of NSG mice. Additionally, we describe a way to manufacture high density BTTs which are ci opreservable, this would greatly ease supply chain constraints on any potential treatment.

[0165] Primary human hepatocytes (PHHs) and mesenchymal stem cells (MSCs) can be aggregated into spheroids and bioprinted three-dimensionally into a tissue therapeutic. The tissue therapeutic can be evaluated by biomarker measurement, such as at one month postimplantation after subcutaneous or intraperitoneal implantation in mice, with live / dead analysis.

[0166] Specifically, PHHs and MSCs were co-aggregated into spheroids which were then suspended in an alginate-based biomaterial. Tissue therapeutics containing 1 x 106PHHs and 1 x 106MSCs or a cell-free control were generated and then implanted into either the IP or SQ space of immune-deficient NSG mice. Blood was collected over a month post-implantation and biomarkers of liver function were measured including human albumin. Follow-up experiments were performed without pre-aggregation of the cells, instead tissue therapeutics were bioprinted using singularized cells which were capable of aggregation after printing as a subset of these fibers were cryopreserved and thawed to demonstrate long-term storage capacity.

[0167] Tissue therapeutics were removed from the NSG mice after 28 days. A calcein AM / propidium iodide viability assay was performed to determine cell viability in the in vitro control tissue therapeutics and the retrieved ex vivo tissue therapeutics. The results are shown in FIG. 4A which demonstrate that the cells retain high viability when implanted either IP or SQ into NSG mice as compared to incubator-maintained controls.

[0168] FIG. 4B shows the plasma albumin concentration (via ELISA) for NSG mice that had the tissue therapeutics with PHH or PHH and MSC implanted IP. These results demonstrate that tissue therapeutics which incorporate MSCs have a much greater longevity of function after implantation compared to tissue therapeutics that do not include MSCs.

[0169] FIG. 4C shows the surgical wounds (as indicated by black arrows) for IP and SQ implantation in NSG mice 3 days post-implantation. In either case, the wound closed, however, it was much smaller when in the mouse with SQ implantation. The outline of the tissue therapeutic can be seen beneath the skin of the SQ mouse.

[0170] FIG. 4D shows the plasma albumin concentration for NSG mice implanted with the tissue therapeutics either IP or SQ and demonstrates similar levels of plasma albumin as between IP and SQ implantation, suggesting there is an equivalence of function between the two implantation sites.

[0171] SQ surgeries were comparatively non-invasive. These results demonstrate that minimally invasive surgery for implantation of liver cell therapies may have several benefits. The larger space for SQ implantation could allow for the implantation of larger tissue therapeutics, as well as for more frequent monitoring and replacement. Decreased invasiveness may also make the procedure an option for non-life-threatening diseases, or in patients who may not tolerate surgery. The data shows that the use of thin fibers in tissue therapeutic manufacturing can improve cell density and allow for the creation of smaller, more functional therapeutics. This new manufacturing methodology also has the potential to allow for cry opreservation of BTTs which would greatly improve the ability to quickly provide treatments when patients become sick. This is especially relevant to conditions like ALF where the time between disease onset and death is limited.Example 4. Cryopreservation of Tissue Fibers Printed with HepG2 Cells or PHH / MSCs (3:1 ratio)

[0172] This Example demonstrates successful cryopreservation, thawing and aggregation of cells in a bioprinted tissue fiber containing dissociated single HepG2 cells. Core-shell fibers areused as a representative example in this particular Example, but the concept is applicable to other fibers, for example annulus fibers.

[0173] A 2% SLG-100 alginate solution was prepared by diluting lyophilized powder in saline. The cross-linker material consisted of an aqueous solution of 95 mM CaCh, 2 mM BaCk, and 20% w / w PEG (8 kDa).

[0174] Core biomaterial was prepared by thawing HepG2 cells and pelleting them in a centrifuge. The cell pellet was diluted to 75% with a 20% (w / w) solution of high molecular weight fish gelatin at 37 °C.

[0175] Core-shell fibers were printed using an Aspect Biosystems RX1 printer. A two-layer core-shell fiber was produced with an outer shell comprising 2% SLG-100 alginate and the core comprising the cell-containing biomaterial containing dissociated cells. In order to allow for oxygen and nutrients to diffuse into all cells, the core of the fiber (shown with HepG2 cells) was 170 pm in diameter as shown in FIG. 5 A. FIG. 5B shows fluorescent staining for live and dead cells for HepG2’s.

[0176] The fibers could be held for up to 2 hours prior to cryopreservation or allowed to culture and aggregate into tube-shaped spheroid-like aggregates. The printed fibers for cryopreservation were transferred into a cryovial with the addition of cryoprotectant (Cryostor 10). To allow for the time required for the cryoprotectant to diffuse throughout the fibre the vial is incubated at room temperature for 10 minutes. The HepG2 cells were then frozen using a Mr. Frosty freezing container inside a -80 °C freezer. After 16 hours the cells were then transferred to a vapor phase liquid nitrogen tank and kept for at least 2 days.

[0177] For thawing, the fibers were thawed by removing the vials from liquid nitrogen and then immediately (<30 seconds) transferring the vial to a ThawStar cryovial thawing instrument. This device will rapidly warm the vial to the point that the vial is approximately 50% frozen and 50% thawed. After the cycle is complete the vial is transferred to a BSC and using sterile technique the liquid cryoprotectant is removed and then warm 37oC cell culture media (EMEM with 10% FBS) is added. The fibre is then transferred to a cell culture plate with fresh culture media. The fibre is allowed to incubate for 72 hours after which the cells within the core of the fibre will begin to aggregate (Figures 6A-6B). The thawed fibres maintain high viability during the thawing process.

[0178] FIGS. 6A-6B show live / dead fluorescent staining for a HepG2 solid fiber 3 days postthawing (FIG. 6A) and a HepG2 non-cryopreserved control solid fiber (FIG. 6B).Example 5. Cryopreservation of Tissue Fibers Printed With PHH / MSCs

[0179] This Example demonstrates successful cryopreservation, thawing and aggregation of cells in a bioprinted tissue fiber containing dissociated single primary human hepatocytes (PHH) and mesenchymal stem cells (MSCs) in a 3:1 ratio.Methods

[0180] A 2% SLG-100 alginate solution was prepared by diluting lyophilized powder in saline. The cross-linker material consisted of an aqueous solution of 95 mM CaCh, 2 mM BaCk, and 5% w / w PVA.

[0181] Single dissociated primary human hepatocytes (PHH) with mesenchymal stem cells (MSCs) (in a 3: 1 ratio) were suspended in a 20% (w / w) solution of high molecular weight fish gelatin at a volume:volume ratio of 3: 1 (to a final 5% gelatin concentration).

[0182] Core biomaterial was prepared by thawing cells and pelleting them in a centrifuge. The cell pellet was diluted to 75% with a 20% (w / w) solution of fish gelatin at 37 °C. HepG2 cells or a ratio of 3: 1 primary human hepatocytes (PHH) and mesenchymal stem cells (MSCs) were prepared.

[0183] Core-shell fibers were printed using an Aspect Biosystems RX1 printer with flow rates of 40 core, 100 shell and 70 cross-linker. The nozzle of the printhead was placed into a bath of 125 mM CaCh dissolved in water. A two-layer core-shell fiber was produced with an outer shell comprising 2% SLG-100 alginate and the core comprising the cell-containing biomaterial containing dissociated cells.

[0184] Some fibers were cultured using standard culture conditions and some were frozen. Frozen fibers were placed in cryovials filled with NG5A Cryopreserv and cooled by 10 °C per min until the chamber is -90 °C, held until the sample is removed; and moved to LN2 vapour. Fibers were thawed after more than 3 days and cultured using standard conditions. Briefly, fibers were cultured in William’s E Medium supplemented with 10% FBS, lx ITS and lOuM Dexamethasone. After 1 day the media was changed to William's E Medium with lx ITS and 10um Dexamethasone. To thaw, the fibers were thawed by removing the vials from liquid nitrogen and then immediately (<30 seconds) transferring the vial to a ThawStar cryovial thawing instrument. This device will rapidly warm the vial to the point that the vial is approximately 50% frozen and 50% thawed. After the cycle is complete the vial is transferred to a BSC and using sterile technique the liquid cryoprotectant is removed and then warm 37oC cell culture media is added. The fibre is then transferred to a cell culture plate with fresh culture media. The fibre is incubated in the media for 30 seconds and then replaced with fresh media. Culture media was William’s E Medium supplemented with 10% FBS, lx ITS and I OUM Dexamethasone. After 1 day the media was changed to William's E Medium with lx ITS and 10 u Dexamethasone. Cyp3 A4 activity was analyzed using a Promega P450 Gio assay and albumin was calculated using an ELISA. These fibers were compared to traditionally printed fibers that contained hepatocytes that had been aggregated ahead of time before printing where the fiber was a coreshell fiber with a wider diameter (1000 pm) and a core material of 1.5% alginate and shell of 2% alginate.Results[00185J Solid fibers were found to aggregate after printing as observed by both brightfield microscopy as well as formalin fixing, paraffin embedding, (FFPE) followed by H&E staining. Cells were seen to aggregate after 3 days. Non-frozen cells were cultured and allowed to aggregate (Figure 7). FIG. 7 shows the functional assay results for Cyp3A4 and the results of the albumin production assay comparing solid fibers at days 3 and 5 compared to traditional (preaggregated spheroid fibers). Functional assays found that solid fibers had higher function in terms of albumin production over 5 days. On day 1, albumin production was 4800 ng / mL for pre-aggregated fibers compared to 13,000 ng / mL for solid fibers. Cyp3A4 activity was also 6.3- fold higher in solid fibers compared to pre-aggregated fibers. After cryopreservation and thawing, the albumin production after cryopreservation was between 70% and 86% that of noncryopreserved fibers for 1 week post-thawing. These cryopreserved fibers were also able to aggregate post-thaw as determined by brightfield imaging.Conclusion

[0186] Three main objectives were accomplished. First, it was demonstrated that cells can be packed more tightly than previous expected while having post-printing aggregation.Secondly, fibers produced in this Example were shown to have higher function than fibers printed using pre-aggregated cells. Thirdly, the fibers were shown to be cryopreservable with minimal loss of function. In combination, these elements greatly improve the feasibility of manufacturing a successful off-the-shelf allogeneic cell therapy for liver disease. Other therapeutic modalities can include xenogeneic and autologous therapies.Example 6. Cryopreservation of Tissue Fibers Printed With PHH / MSCs or Adrenocortical Cells

[0187] This Example demonstrates successful cry opreservation, thawing and aggregation of cells in a bioprinted tissue fiber containing dissociated single primary human hepatocytes (PHH) and mesenchymal stem cells (MSCs) in a 3: 1 ratio or dissociated primary human adrenocortical cells as well as the effect of using pre-aggregated cells for bioprinting.

[0188] Single cells and aggregates of either PHH / MSCs or adrenocortical cells were bioprinted in solid fiber (dissociated cell) and pre-aggregated annulus formats, respectively. FIGS. 8A-8C and 9 show representative images of solid fibers containing PHH+MSCs or adrenal cells (FIG. 8A), cell aggregates in an annulus layer (FIG. 8B), a cross-sectional view of bioprinted annulus fibers with a cell-free inner core, cell-containing annulus layer and cell-free outer shell (FIG. 8C), and a representative image of a solid fiber containing single cells in the annulus layer (FIG. 9) where annotations indicate dimensions of outer shell (-71.6-78.5 pm), cell-containing annulus (-270.4-292.7 pm), cell-free core (-424.8 pm) and total fiber diameter (-1.19 mm).

[0189] Fresh fibers were maintained in media at 37 °C and 5% CO2. Cryopreserved fibers were cryopreserved using a control-rate freezer and held in liquid nitrogen for 72 hours before being thawed. To assess viability, fibers were stained with Calcein-AM (green) and propidium iodide (red) (FIG. 10A; FIG. 10B).

[0190] Solid fibers (containing dissociated cells) were cultured in William’s E media with FBS, ITS and dexamethasone for hepatocytes and then switched to media without FBS. Preaggregated fibers were cultured in media without FBS. Media was collected 24 hours following bioprinting or thawing to quantify albumin. (Secreted albumin from conditioned media was quantified by ELISA (Abeam catalog # ab235642 or ab 179887) according to the manufacturer’s instructions. 24-hour conditioned media was also quantified by ELISA for cortisol secretion forsolid fibers containing adrenocortical cells (Abeam catalog # ab 108665), and the adrenocortical cell media was Human Adrenal Cortical Cell Culture Medium (iXCells Biotechnologies).Results

[0191] FIGS. 10A-10B show fluorescent staining for live and dead cells for the solid fiber and aggregate annulus formats.

[0192] FIGS. 11A-12B show albumin and cortisol secretion (in ng / million cells / day for FIGS. 11 A-l IB and relative secretion for FIGS. 12A-12B) for solid fibers or aggregates (fresh or cryopreserved) printed with PHH + MSCs (FIGS. 11 A and 12A, for albumin) or adrenocortical cells (FIGS. 1 IB and 12B, for cortisol).Conclusion

[0193] Combined this data suggests that the solid fiber and solid annulus fiber method of bioprinting allows for cry opreservation with much greater function and viability upon thawing when compared to freezing of bioprinted cell aggregates.

[0194] The embodiments and examples described above are intended to be merely illustrative and non-limiting. Those skilled in the art will recognize or will be able to ascertain using no more than routine experimentation, numerous equivalents of specific compounds, materials and procedures. All such equivalents are considered to be within the scope and are encompassed by the appended claims.

Claims

CLAIMS:What is claimed is:

1. A cryopreserved tissue fiber comprising a continuously bioprinted core / shell fiber encapsulating a cell population for implantation, wherein said core / shell fiber comprises a core and at least one shell, wherein the cell population comprises at least 90%, at least 95%, at least 98%, at least 99%, or 100% dissociated cells, embedded in a biocompatible matrix, and wherein the fiber is cryopreserved prior to aggregation of the dissociated cells.

2. The cryopreserved tissue fiber of claim 1, wherein the cell population is encapsulated in the core and / or in the at least one shell.

3. The cryopreserved tissue fiber of claim 2, wherein the core / shell fiber comprises at least two shells, the innermost shell comprising an annulus layer encapsulating the cell population.

4. The cryopreserved tissue fiber of any preceding claim, wherein the biocompatible matrix does not include DMSO, preferably wherein the core / shell fiber is bioprinted without including DMSO in the core or in the at least one shell.

5. The cryopreserved tissue fiber of any preceding claim, wherein the biocompatible matrix further comprises a viscosifier, preferably wherein said viscosifier comprises fish gelatin.

6. The cryopreserved tissue fiber any preceding claim, wherein the core and the at least one shell comprise the same hydrogel material; preferably wherein the hydrogel material is alginate.

7. The cryopreserved tissue fiber of claim 6, wherein the core and / or the at least one shell comprises a chemically modified alginate.

8. The cryopreserved tissue fiber of any preceding claim, wherein the biocompatible matrix comprises at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, or at least about 80% v / v of cells in physiological matrix material.

9. The cryopreserved tissue fiber of claim 8, wherein the fiber is a high-density fiber and the biocompatible matrix comprises at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, or at least about 85% v / v of cells in physiological matrix material.

10. The cryopreserved tissue fiber of any preceding claim, wherein the biocompatible matrix comprises a ratio of cells : physiological matrix material of at least about 2: 1, preferably at least about 3: 1, more preferably at least about 4:1.

11. The cryopreserved tissue fiber of claim 2, wherein the cell population is encapsulated in the core, and the core diameter is between about 100 um and about 200 um, preferably between about 130 um and about 200 um, preferably between about 140 um and about 190 um, more preferably between about 150 um to 175 um, more preferably about 150 um, about 155 um, about 160 um, about 165 um, about 170 um, or about 175 um.

12. The cryopreserved tissue fiber of claim 11, wherein the shell thickness is between about 30 um and 80 um, preferably between about 50 um and 70 um, more preferably about 60 um, about 65 um, or about 70 um.

13. The cryopreserved tissue fiber of claim 11 or 12, wherein the total fiber diameter is between about 270 um and about 330 um, preferably between about 280 um and about 320 um,more preferably about 290 um to about 310 um, more preferably about 290 um, about 300 um, or about 310 um.

14. The cryopreserved tissue fiber of claim 3, wherein the cell population is encapsulated in the annulus layer, and the core diameter is between about 300 um and about 500 um, preferably between about 350 um and about 450 um, more preferably about 350 um, about 375 um, about 400 um, about 425 um, or about 450 um.

15. The cryopreserved tissue fiber of claim 14, wherein the annulus layer thickness is between about 100 um and about 300 um, preferably between about 130 um and about 200 um, preferably between about 140 um and about 190 um, more preferably between about 150 um to 175 um, more preferably about 150 um, about 155 um, about 160 um, about 165 um, about 170 um, or about 175 um.

16. The cryopreserved fiber of claim 14 or 15, further comprising an outer shell having a thickness between about 30 um and 80 um, more preferably between about 45 um and 70 um, more preferably about 60 um, about 65 um, or about 70 um.

17. The cryopreserved tissue fiber of any one of claims 14 - 16, wherein the total fiber diameter is between about 500-1200 um, preferably between about 700 um and about 1000 um, more preferably about 800 um, about 900 um, or about 1000 um.

18. The cryopreserved tissue fiber of any preceding claim, wherein the cell population comprises primary cells from endocrine and exocrine glands selected from the group consisting of pancreas, liver, kidney, parathyroid, thyroid, pineal gland, pituitary gland, thymus, adrenal gland, ovary, testis, tonsillar, enteroendocrine cells, stem cells, stem-cell derived cells of any of the foregoing primary cell types, or cells engineered to secrete a therapeutic agent.

19. The cryopreserved tissue fiber of claim 18, wherein the cell population comprises HepG2s, primary human hepatocytes (PHHs), mesenchymal stem cells (MSCs), embryonic stem cell- or induced pluripotent stem cell-derived hepatocyte-like cells, liver stem cell-derived hepatocyte-like cells, xenogeneic hepatocytes, genetically engineered stem cell-derived hepatocytes, stem-derived or primary endothelial cells, and combinations thereof.

20. The cryopreserved fiber of claim 19, wherein the dissociated cells comprise a 3: 1 ratio of PHHs and MSCs.

21. A composition / medical device for implanting a cell population into a subject in need thereof, comprising a multilayer structure comprising a tissue fiber according to any one of claims 1-20, wherein the composition is cryopreserved prior to aggregation of the dissociated cells.

22. The composition / device of claim 21, wherein the multilayer structure comprises at least one conformal coating.

23. The composition / device of claim 22 wherein the multilayer structure comprises a first inner coating and a second outer coating; preferably wherein the first inner coating comprises a hydrogel having a material strength greater than that of the second outer coating.

24. The composition / device of any one of claims 21-23 wherein the core and / or the at least one shell is compartmentalized along the length of the fiber.

25. A method of implanting a cell population into a subject in need thereof, comprising: a. thawing a cryopreserved tissue fiber according to any one of claims 1-20 or a composition / device according to any one of claims 21-24; andC. implanting said fiber or said composition / device into said subject.

26. The method of claim 25, further comprising culturing said tissue fiber or said composition / device in fresh culture media after thawing until the dissociated cells form aggregates in said tissue fiber; prior to implantation into said subject.

27. A method of making the tissue fiber of any one of claims 1-20, comprising the steps of a. providing a bioprinting system comprising a print head comprising a plurality of microfluidic channels to selectively provide a core material, at least one shell material, and a sheath fluid to a dispensing orifice; a receiving surface for receiving the materials dispensed from the orifice, a positioning unit for positioning the orifice of the print head in three dimensional space, the positioning unit operably coupled to the print head, and at least one dispensing means for dispensing the fiber from the dispensing orifice; b. via the bioprinting system, simultaneously printing the core and the at least one shell of the tissue fiber, wherein the cell population is encapsulated in the core and / or in the at least one shell; and c. cry opreserving said bioprinted tissue fiber with a cryoprotectant; wherein the cell population comprises at least 95% dissociated cells embedded in a biocompatible matrix, and wherein the fiber is cryopreserved prior to aggregation of the dissociated cells; ; preferably within about 10 minutes to an hour of bioprinting, more preferably within about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 or 60 minutes of bioprinting.

28. The method of claim 27 for making a composition / medical device according to any one of claims 2-24, wherein said printing step comprises dispensing the continuously bioprinted core / shell fiber on said receiving surface to form a multilayer structure, and wherein the multilayer structure is cryopreserved prior to aggregation of the dissociated cells in the fiber.

29. The method of claim 27 or 28, wherein the core material and at least one shell material are bioprinted without DMSO.

30. The method of any one of claims 27-29, wherein said cryopreserving step comprises adding cryoprotectant to the printed fiber at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 minutes before freezing.

31. The method of any one of claims 27-30, wherein the core material further comprises a viscosifier, preferably wherein the viscosity of the core material and the shell material are substantially the same.

32. The method of any one of claims 27-31, wherein said receiving surface comprises a fabrication platform for supporting the continuously bioprinted core / shell fiber, the fabrication platform comprising a frame defining a void and comprising a plurality of posts on opposing sides of the frame for securing and suspending the continuously bioprinted fiber during printing; and the printing step comprises dispensing the fiber around a plurality of said posts to form a lattice structure comprising at least two, three, four, or five layers of the fiber.

33. The method of any one of claims 27-32, further comprising coating the lattice structure after printing is completed.

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